Compositions and methods

ABSTRACT

Provided herein are oligomeric compounds with conjugate groups. In certain embodiments, the oligomeric compounds are conjugated to N-Acetylgalactosamine.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0255USC1SEQ_ST25.txt created Jun. 8, 2017, which is 504 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The principle behind antisense technology is that an antisense compoundhybridizes to a target nucleic acid and modulates the amount, activity,and/or function of the target nucleic acid. For example in certaininstances, antisense compounds result in altered transcription ortranslation of a target. Such modulation of expression can be achievedby, for example, target mRNA degradation or occupancy-based inhibition.An example of modulation of RNA target function by degradation is RNaseH-based degradation of the target RNA upon hybridization with a DNA-likeantisense compound. Another example of modulation of gene expression bytarget degradation is RNA interference (RNAi). RNAi refers toantisense-mediated gene silencing through a mechanism that utilizes theRNA-induced silencing complex (RISC). An additional example ofmodulation of RNA target function is by an occupancy-based mechanismsuch as is employed naturally by microRNA. MicroRNAs are smallnon-coding RNAs that regulate the expression of protein-coding RNAs. Thebinding of an antisense compound to a microRNA prevents that microRNAfrom binding to its messenger RNA targets, and thus interferes with thefunction of the microRNA. MicroRNA mimics can enhance native microRNAfunction. Certain antisense compounds alter splicing of pre-mRNA.Regardless of the specific mechanism, sequence-specificity makesantisense compounds attractive as tools for target validation and genefunctionalization, as well as therapeutics to selectively modulate theexpression of genes involved in the pathogenesis of diseases.

Antisense technology is an effective means for modulating the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides may be incorporated intoantisense compounds to enhance one or more properties, such as nucleaseresistance, pharmacokinetics or affinity for a target nucleic acid. In1998, the antisense compound, Vitravene® (fomivirsen; developed by IsisPharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug toachieve marketing clearance from the U.S. Food and Drug Administration(FDA), and is currently a treatment of cytomegalovirus (CMV)-inducedretinitis in AIDS patients.

New chemical modifications have improved the potency and efficacy ofantisense compounds, uncovering the potential for oral delivery as wellas enhancing subcutaneous administration, decreasing potential for sideeffects, and leading to improvements in patient convenience. Chemicalmodifications increasing potency of antisense compounds allowadministration of lower doses, which reduces the potential for toxicity,as well as decreasing overall cost of therapy. Modifications increasingthe resistance to degradation result in slower clearance from the body,allowing for less frequent dosing. Different types of chemicalmodifications can be combined in one compound to further optimize thecompound's efficacy.

SUMMARY OF THE INVENTION

In certain embodiments, the present disclosure provides conjugatedantisense compounds. In certain embodiments, the present disclosureprovides conjugated antisense compounds comprising an antisenseoligonucleotide complementary to a nucleic acid transcript. In certainembodiments, the present disclosure provides methods comprisingcontacting a cell with a conjugated antisense compound comprising anantisense oligonucleotide complementary to a nucleic acid transcript. Incertain embodiments, the present disclosure provides methods comprisingcontacting a cell with a conjugated antisense compound comprising anantisense oligonucleotide and reducing the amount or activity of anucleic acid transcript in a cell.

The asialoglycoprotein receptor (ASGP-R) has been described previously.See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005).Such receptors are expressed on liver cells, particularly hepatocytes.Further, it has been shown that compounds comprising clusters of threeN-acetylgalactosamine (GalNAc) ligands are capable of binding to theASGP-R, resulting in uptake of the compound into the cell. See e.g.,Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231(May 2008). Accordingly, conjugates comprising such GalNAc clusters havebeen used to facilitate uptake of certain compounds into liver cells,specifically hepatocytes. For example it has been shown that certainGalNAc-containing conjugates increase activity of duplex siRNA compoundsin liver cells in vivo. In such instances, the GalNAc-containingconjugate is typically attached to the sense strand of the siRNA duplex.Since the sense strand is discarded before the antisense strandultimately hybridizes with the target nucleic acid, there is littleconcern that the conjugate will interfere with activity. Typically, theconjugate is attached to the 3′ end of the sense strand of the siRNA.See e.g., U.S. Pat. No. 8,106,022. Certain conjugate groups describedherein are more active and/or easier to synthesize than conjugate groupspreviously described.

In certain embodiments of the present invention, conjugates are attachedto single-stranded antisense compounds, including, but not limited toRNase H based antisense compounds and antisense compounds that altersplicing of a pre-mRNA target nucleic acid. In such embodiments, theconjugate should remain attached to the antisense compound long enoughto provide benefit (improved uptake into cells) but then should eitherbe cleaved, or otherwise not interfere with the subsequent stepsnecessary for activity, such as hybridization to a target nucleic acidand interaction with RNase H or enzymes associated with splicing orsplice modulation. This balance of properties is more important in thesetting of single-stranded antisense compounds than in siRNA compounds,where the conjugate may simply be attached to the sense strand.Disclosed herein are conjugated single-stranded antisense compoundshaving improved potency in liver cells in vivo compared with the sameantisense compound lacking the conjugate. Given the required balance ofproperties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a cleavablemoiety. As noted, without wishing to be bound by mechanism, it islogical that the conjugate should remain on the compound long enough toprovide enhancement in uptake, but after that, it is desirable for someportion or, ideally, all of the conjugate to be cleaved, releasing theparent compound (e.g., antisense compound) in its most active form. Incertain embodiments, the cleavable moiety is a cleavable nucleoside.Such embodiments take advantage of endogenous nucleases in the cell byattaching the rest of the conjugate (the cluster) to the antisenseoligonucleotide through a nucleoside via one or more cleavable bonds,such as those of a phosphodiester linkage. In certain embodiments, thecluster is bound to the cleavable nucleoside through a phosphodiesterlinkage. In certain embodiments, the cleavable nucleoside is attached tothe antisense oligonucleotide (antisense compound) by a phosphodiesterlinkage. In certain embodiments, the conjugate group may comprise two orthree cleavable nucleosides. In such embodiments, such cleavablenucleosides are linked to one another, to the antisense compound and/orto the cluster via cleavable bonds (such as those of a phosphodiesterlinkage). Certain conjugates herein do not comprise a cleavablenucleoside and instead comprise a cleavable bond. It is shown that thatsufficient cleavage of the conjugate from the oligonucleotide isprovided by at least one bond that is vulnerable to cleavage in the cell(a cleavable bond).

In certain embodiments, conjugated antisense compounds are prodrugs.Such prodrugs are administered to an animal and are ultimatelymetabolized to a more active form. For example, conjugated antisensecompounds are cleaved to remove all or part of the conjugate resultingin the active (or more active) form of the antisense compound lackingall or some of the conjugate.

In certain embodiments, conjugates are attached at the 5′ end of anoligonucleotide. Certain such 5′-conjugates are cleaved more efficientlythan counterparts having a similar conjugate group attached at the 3′end. In certain embodiments, improved activity may correlate withimproved cleavage. In certain embodiments, oligonucleotides comprising aconjugate at the 5′ end have greater efficacy than oligonucleotidescomprising a conjugate at the 3′ end (see, for example, Examples 56, 81,83, and 84). Further, 5′-attachment allows simpler oligonucleotidesynthesis. Typically, oligonucleotides are synthesized on a solidsupport in the 3′ to 5′ direction. To make a 3′-conjugatedoligonucleotide, typically one attaches a pre-conjugated 3′ nucleosideto the solid support and then builds the oligonucleotide as usual.However, attaching that conjugated nucleoside to the solid support addscomplication to the synthesis. Further, using that approach, theconjugate is then present throughout the synthesis of theoligonucleotide and can become degraded during subsequent steps or maylimit the sorts of reactions and reagents that can be used. Using thestructures and techniques described herein for 5′-conjugatedoligonucleotides, one can synthesize the oligonucleotide using standardautomated techniques and introduce the conjugate with the final(5′-most) nucleoside or after the oligonucleotide has been cleaved fromthe solid support.

In view of the art and the present disclosure, one of ordinary skill caneasily make any of the conjugates and conjugated oligonucleotidesherein. Moreover, synthesis of certain such conjugates and conjugatedoligonucleotides disclosed herein is easier and/or requires few steps,and is therefore less expensive than that of conjugates previouslydisclosed, providing advantages in manufacturing. For example, thesynthesis of certain conjugate groups consists of fewer synthetic steps,resulting in increased yield, relative to conjugate groups previouslydescribed. Conjugate groups such as GalNAc3-10 in Example 46 andGalNAc3-7 in Example 48 are much simpler than previously describedconjugates such as those described in U.S. Pat. No. 8,106,022 or U.S.Pat. No. 7,262,177 that require assembly of more chemical intermediates.Accordingly, these and other conjugates described herein have advantagesover previously described compounds for use with any oligonucleotide,including single-stranded oligonucleotides and either strand ofdouble-stranded oligonucleotides (e.g., siRNA).

Similarly, disclosed herein are conjugate groups having only one or twoGalNAc ligands. As shown, such conjugates groups improve activity ofantisense compounds. Such compounds are much easier to prepare thanconjugates comprising three GalNAc ligands. Conjugate groups comprisingone or two GalNAc ligands may be attached to any antisense compounds,including single-stranded oligonucleotides and either strand ofdouble-stranded oligonucleotides (e.g., siRNA).

In certain embodiments, the conjugates herein do not substantially altercertain measures of tolerability. For example, it is shown herein thatconjugated antisense compounds are not more immunogenic thanunconjugated parent compounds. Since potency is improved, embodiments inwhich tolerability remains the same (or indeed even if tolerabilityworsens only slightly compared to the gains in potency) have improvedproperties for therapy.

In certain embodiments, conjugation allows one to alter antisensecompounds in ways that have less attractive consequences in the absenceof conjugation. For example, in certain embodiments, replacing one ormore phosphorothioate linkages of a fully phosphorothioate antisensecompound with phosphodiester linkages results in improvement in somemeasures of tolerability. For example, in certain instances, suchantisense compounds having one or more phosphodiester are lessimmunogenic than the same compound in which each linkage is aphosphorothioate. However, in certain instances, as shown in Example 26,that same replacement of one or more phosphorothioate linkages withphosphodiester linkages also results in reduced cellular uptake and/orloss in potency. In certain embodiments, conjugated antisense compoundsdescribed herein tolerate such change in linkages with little or no lossin uptake and potency when compared to the conjugatedfull-phosphorothioate counterpart. In fact, in certain embodiments, forexample, in Examples 44, 57, 59, and 86, oligonucleotides comprising aconjugate and at least one phosphodiester internucleoside linkageactually exhibit increased potency in vivo even relative to a fullphosphorothioate counterpart also comprising the same conjugate.Moreover, since conjugation results in substantial increases inuptake/potency a small loss in that substantial gain may be acceptableto achieve improved tolerability. Accordingly, in certain embodiments,conjugated antisense compounds comprise at least one phosphodiesterlinkage.

In certain embodiments, conjugation of antisense compounds hereinresults in increased delivery, uptake and activity in hepatocytes. Thus,more compound is delivered to liver tissue. However, in certainembodiments, that increased delivery alone does not explain the entireincrease in activity. In certain such embodiments, more compound entershepatocytes. In certain embodiments, even that increased hepatocyteuptake does not explain the entire increase in activity. In suchembodiments, productive uptake of the conjugated compound is increased.For example, as shown in Example 102, certain embodiments ofGalNAc-containing conjugates increase enrichment of antisenseoligonucleotides in hepatocytes versus non-parenchymal cells. Thisenrichment is beneficial for oligonucleotides that target genes that areexpressed in hepatocytes.

In certain embodiments, conjugated antisense compounds herein result inreduced kidney exposure. For example, as shown in Example 20, theconcentrations of antisense oligonucleotides comprising certainembodiments of GalNAc-containing conjugates are lower in the kidney thanthat of antisense oligonucleotides lacking a GalNAc-containingconjugate. This has several beneficial therapeutic implications. Fortherapeutic indications where activity in the kidney is not sought,exposure to kidney risks kidney toxicity without corresponding benefit.Moreover, high concentration in kidney typically results in loss ofcompound to the urine resulting in faster clearance. Accordingly fornon-kidney targets, kidney accumulation is undesired.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the formula:

A-B-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the branching group“D” branches as many times as is necessary to accommodate the number of(E-F) groups as indicated by “q”. Thus, where q=1, the formula is:

A-B-C-D-E-F

where q=2, the formula is:

where q=3, the formula is:

where q=4, the formula is:

where q=5, the formula is:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

The present disclosure provides the following non-limiting numberedembodiments:

Embodiment 1

The conjugated antisense compound of any of embodiments 1179 to 1182,wherein the tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

Embodiment 2

The conjugated antisense compound of any of embodiments 1179 to 1182,wherein the tether has the structure:

Embodiment 3

The conjugated antisense compound of any of embodiments 1179 to 1182 or1688 to 1689, wherein the linker has a structure selected from among:

Embodiment 4

The conjugated antisense compound of any of embodiments 1179 to 1182 or1688 to 1689, wherein the linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

Embodiment 5

The conjugated antisense compound of any of embodiments 1179 to 1182 or1688 to 1689, wherein the linker has the structure:

In embodiments having more than one of a particular variable (e.g., morethan one “m” or “n”), unless otherwise indicated, each such particularvariable is selected independently. Thus, for a structure having morethan one n, each n is selected independently, so they may or may not bethe same as one another.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the disclosure. Herein, the use of thesingular includes the plural unless specifically stated otherwise. Asused herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

A. Definitions

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “2′-F nucleoside” refers to a nucleoside comprising asugar comprising fluorine at the 2′ position. Unless otherwiseindicated, the fluorine in a 2′-F nucleoside is in the ribo position(replacing the OH of a natural ribose).

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification of the 2′ position of a furosyl ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

“3′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 3′-most nucleotide of a particular antisensecompound.

“5′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 5′-most nucleotide of a particular antisensecompound.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“About” means within ±10% of a value. For example, if it is stated, “amarker may be increased by about 50%”, it is implied that the marker maybe increased between 45%-55%.

“Active pharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual. For example, in certain embodiments anantisense oligonucleotide targeted to PTP1B is an active pharmaceuticalagent.

“Active target region” or “target region” means a region to which one ormore active antisense compounds is targeted. “Active antisensecompounds” means antisense compounds that reduce target nucleic acidlevels or protein levels.

“Adipogenesis” means the development of fat cells from preadipocytes.“Lipogenesis” means the production or formation of fat, either fattydegeneration or fatty infiltration.

“Adiposity” or “Obesity” refers to the state of being obese or anexcessively high amount of body fat or adipose tissue in relation tolean body mass. The amount of body fat includes concern for both thedistribution of fat throughout the body and the size and mass of theadipose tissue deposits. Body fat distribution can be estimated byskin-fold measures, waist-to-hip circumference ratios, or techniquessuch as ultrasound, computed tomography, or magnetic resonance imaging.According to the Center for Disease Control and Prevention, individualswith a body mass index (BMI) of 30 or more are considered obese. Theterm “Obesity” as used herein includes conditions where there is anincrease in body fat beyond the physical requirement as a result ofexcess accumulation of adipose tissue in the body. The term “obesity”includes, but is not limited to, the following conditions: adult-onsetobesity; alimentary obesity; endogenous or inflammatory obesity;endocrine obesity; familial obesity; hyperinsulinar obesity;hyperplastic-hypertrophic obesity; hypogonadal obesity; hypothyroidobesity; lifelong obesity; morbid obesity and exogenous obesity.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Administering” means providing an agent to an animal, and includes, butis not limited to, administering by a medical professional andself-administering.

“Agent” means an active substance that can provide a therapeutic benefitwhen administered to an animal. “First Agent” means a therapeuticcompound provided herein. For example, a first agent can be an antisenseoligonucleotide targeting PTP1B. “Second agent” means a secondtherapeutic compound of the invention (e.g. a second antisenseoligonucleotide targeting PTP1B) and/or a non-PTP1B therapeuticcompound.

“Amelioration” refers to a lessening of at least one indicator, sign, orsymptom of an associated disease, disorder, or condition. The severityof indicators can be determined by subjective or objective measures,which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding.

“Antisense inhibition” means reduction of target nucleic acid levels ortarget protein levels in the presence of an antisense compoundcomplementary to a target nucleic acid compared to target nucleic acidlevels or target protein levels in the absence of the antisensecompound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid.

“Bicyclic sugar” means a furosyl ring modified by the bridging of twonon-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside or nucleotide includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleotides is chemically distinct from a region having nucleotideswithout 2′-O-methoxyethyl modifications.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Chemicalmodifications of oligonucleotides include nucleoside modifications(including sugar moiety modifications and nucleobase modifications) andinternucleoside linkage modifications. In reference to anoligonucleotide, chemical modification does not include differences onlyin nucleobase sequence.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions.

“Co-administration” means administration of two or more agents to anindividual. The two or more agents can be in a single pharmaceuticalcomposition, or can be in separate pharmaceutical compositions. Each ofthe two or more agents can be administered through the same or differentroutes of administration. Co-administration encompasses parallel orsequential administration.

“Cholesterol” is a sterol molecule found in the cell membranes of allanimal tissues. Cholesterol must be transported in an animal's bloodplasma by lipoproteins including very low density lipoprotein (VLDL),intermediate density lipoprotein (IDL), low density lipoprotein (LDL),and high density lipoprotein (HDL). “Plasma cholesterol” refers to thesum of all lipoproteins (VDL, IDL, LDL, HDL) esterified and/ornon-esterified cholesterol present in the plasma or serum.

“Cholesterol absorption inhibitor” means an agent that inhibits theabsorption of exogenous cholesterol obtained from diet.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′position of the sugar portion of the nucleotide. Deoxyribonucleotidesmay be modified with any of a variety of substituents.

“Diabetes mellitus” or “diabetes” is a syndrome characterized bydisordered metabolism and abnormally high blood sugar (hyperglycemia)resulting from insufficient levels of insulin or reduced insulinsensitivity. The characteristic symptoms are excessive urine production(polyuria) due to high blood glucose levels, excessive thirst andincreased fluid intake (polydipsia) attempting to compensate forincreased urination, blurred vision due to high blood glucose effects onthe eye's optics, unexplained weight loss, and lethargy.

“Diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means acondition characterized by Type 2 diabetes, reduced HDL-C, elevatedtriglycerides, and elevated small, dense LDL particles.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition can be aliquid, e.g. saline solution.

“Dyslipidemia” refers to a disorder of lipid and/or lipoproteinmetabolism, including lipid and/or lipoprotein overproduction ordeficiency. Dyslipidemias may be manifested by elevation of lipids suchas cholesterol and triglycerides as well as lipoproteins such aslow-density lipoprotein (LDL) cholesterol.

“Dosage unit” means a form in which a pharmaceutical agent is provided,e.g. pill, tablet, or other dosage unit known in the art. In certainembodiments, a dosage unit is a vial containing lyophilized antisenseoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted antisense oligonucleotide.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose can be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections can be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses can be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” or “therapeutically effective amount” means theamount of active pharmaceutical agent sufficient to effectuate a desiredphysiological outcome in an individual in need of the agent. Theeffective amount can vary among individuals depending on the health andphysical condition of the individual to be treated, the taxonomic groupof the individuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

“Fully complementary” or “100% complementary” means each nucleobase of anucleobase sequence of a first nucleic acid has a complementarynucleobase in a second nucleobase sequence of a second nucleic acid. Incertain embodiments, a first nucleic acid is an antisense compound and atarget nucleic acid is a second nucleic acid.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region can be referred to as a “gap segment” andthe external regions can be referred to as “wing segments.”

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleosides.

“Glucose” is a monosaccharide used by cells as a source of energy andinflammatory intermediate. “Plasma glucose” refers to glucose present inthe plasma.

“HMG-CoA reductase inhibitor” means an agent that acts through theinhibition of the enzyme HMG-CoA reductase, such as atorvastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude an antisense compound and a target nucleic acid.

“Hyperlipidemia” or “hyperlipemia” is a condition characterized byelevated serum lipids or circulating (plasma) lipids. This conditionmanifests an abnormally high concentration of fats. The lipid fractionsin the circulating blood are cholesterol, low density lipoproteins, verylow density lipoproteins and triglycerides.

“Hypertriglyceridemia” means a condition characterized by elevatedtriglyceride levels.

“Identifying” or “selecting an animal with metabolic” means identifyingor selecting a subject having been diagnosed with a metabolic disease,or a metabolic disorder; or, identifying or selecting a subject havingany symptom of a metabolic disease, including, but not limited to,metabolic syndrome, hyperglycemia, hypertriglyceridemia, hypertensionincreased insulin resistance, decreased insulin sensitivity, abovenormal body weight, and/or above normal body fat or any combinationthereof. Such identification may be accomplished by any method,including but not limited to, standard clinical tests or assessments,such as measuring serum or circulating (plasma) blood-glucose, measuringserum or circulating (plasma) triglycerides, measuring blood-pressure,measuring body fat, measuring body weight, and the like.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” or “subject” or “animal” means a human or non-human animalselected for treatment or therapy.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity of a RNA or protein and does notnecessarily indicate a total elimination of expression or activity.

“Insulin resistance” is defined as the condition in which normal amountsof insulin are inadequate to produce a normal insulin response from fat,muscle and liver cells. Insulin resistance in fat cells results inhydrolysis of stored triglycerides, which elevates free fatty acids inthe blood plasma. Insulin resistance in muscle reduces glucose uptakewhereas insulin resistance in liver reduces glucose storage, with botheffects serving to elevate blood glucose. High plasma levels of insulinand glucose due to insulin resistance often leads to metabolic syndromeand type 2 diabetes.

“Insulin sensitivity” is a measure of how effectively an individualprocesses glucose. An individual having high insulin sensitivityeffectively processes glucose whereas an individual with low insulinsensitivity does not effectively process glucose.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Intravenous administration” means administration into a vein.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

“Lipid-lowering therapy” or “lipid lowering agent” means a therapeuticregimen provided to a subject to reduce one or more lipids in a subject.In certain embodiments, a lipid-lowering therapy is provided to reduceone or more of ApoB, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C,triglycerides, small dense LDL particles, and Lp(a) in a subject.Examples of lipid-lowering therapy include statins, fibrates, and MTPinhibitors.

“Major risk factors” refers to factors that contribute to a high riskfor a particular disease or condition. In certain embodiments, majorrisk factors for coronary heart disease include, without limitation,cigarette smoking, hypertension, low HDL-C, family history of coronaryheart disease, age, and other factors disclosed herein.

“Metabolic disease” or “metabolic disorder” refers to a conditioncharacterized by an alteration or disturbance in metabolic function.“Metabolic” and “metabolism” are terms well known in the art andgenerally include the whole range of biochemical processes that occurwithin a living organism. Metabolic diseases or disorders include, butare not limited to, obesity, diabetes, hyperglycemia, prediabetes,non-alcoholic fatty liver disease (NAFLD), metabolic syndrome, insulinresistance, diabetic dyslipidemia, or hypertriglyceridemia or acombination thereof.

“Metabolic syndrome” means a condition characterized by a clustering oflipid and non-lipid cardiovascular risk factors of metabolic origin. Incertain embodiments, metabolic syndrome is identified by the presence ofany 3 of the following factors: waist circumference of greater than 102cm in men or greater than 88 cm in women; serum triglyceride of at least150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL inwomen; blood pressure of at least 130/85 mmHg; and fasting glucose of atleast 110 mg/dL. These determinants can be readily measured in clinicalpractice (JAMA, 2001, 285: 2486-2497).

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Mixed dyslipidemia” means a condition characterized by elevatedcholesterol and elevated triglycerides.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e. aphosphodiester internucleoside bond).

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase”means the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, amodified sugar moiety or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, or modifiednucleobase. A “modified nucleoside” means a nucleoside having,independently, a modified sugar moiety or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified nucleotide.

“Modified sugar” refers to a substitution or change from a naturalsugar.

As used herein, “modified sugar moiety” means a substituted sugar moietyor a sugar surrogate.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“MTP inhibitor” means an agent inhibits the enzyme, microsomaltriglyceride transfer protein.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Non-alcoholic fatty liver disease” or “NAFLD” means a conditioncharacterized by fatty inflammation of the liver that is not due toexcessive alcohol use (for example, alcohol consumption of over 20g/day). In certain embodiments, NAFLD is related to insulin resistanceand the metabolic syndrome. NAFLD encompasses a disease spectrum rangingfrom simple triglyceride accumulation in hepatocytes (hepatic steatosis)to hepatic steatosis with inflammation (steatohepatitis), fibrosis, andcirrhosis.

“Nonalcoholic steatohepatitis” (NASH) occurs from progression of NAFLDbeyond deposition of triglycerides. A “second hit” capable of inducingnecrosis, inflammation, and fibrosis is required for development ofNASH. Candidates for the second-hit can be grouped into broadcategories: factors causing an increase in oxidative stress and factorspromoting expression of proinflammatory cytokines

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids(DNA), single-stranded nucleic acids, double-stranded nucleic acids,small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). Anucleic acid can also comprise a combination of these elements in asingle molecule.

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid. As used herein, “nucleobase” means a group ofatoms that can be linked to a sugar moiety to create a nucleoside thatis capable of incorporation into an oligonucleotide, and wherein thegroup of atoms is capable of bonding with a complementary naturallyoccurring nucleobase of another oligonucleotide or nucleic acid.Nucleobases may be naturally occurring or may be modified.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, or nucleobase modification.

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside. As used herein,“nucleotide” means a nucleoside further comprising a phosphate linkinggroup. As used herein, “linked nucleosides” may or may not be linked byphosphate linkages and thus includes, but is not limited to “linkednucleotides.” As used herein, “linked nucleosides” are nucleosides thatare connected in a continuous sequence (i.e. no additional nucleosidesare present between those that are linked).

“Nucleotide mimetic” includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage).

“Oligomeric compound” or “oligomer” refers to a polymeric structurecomprising two or more sub-structures and capable of hybridizing to aregion of a nucleic acid molecule. In certain embodiments, oligomericcompounds are oligonucleosides. In certain embodiments, oligomericcompounds are oligonucleotides. In certain embodiments, oligomericcompounds are antisense compounds. In certain embodiments, oligomericcompounds are antisense oligonucleotides. In certain embodiments,oligomeric compounds are chimeric oligonucleotides.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intrathecal orintracerebroventricular administration. Administration can becontinuous, or chronic, or short or intermittent.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Peptide refers to polypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeuticbenefit when administered to an individual. For example, in certainembodiments, an antisense oligonucleotide targeted to PTP1B ispharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition can comprise one or more active agents and a sterile aqueoussolution.

“Pharmaceutically acceptable carrier” means a medium or diluent thatdoes not interfere with the structure of the oligonucleotide. Certain,of such carries enable pharmaceutical compositions to be formulated as,for example, tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspension and lozenges for the oral ingestion by a subject.For example, a pharmaceutically acceptable carrier can be a sterileaqueous solution.

“Pharmaceutically acceptable derivative” encompasses pharmaceuticallyacceptable salts, conjugates, prodrugs or isomers of the compoundsdescribed herein.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a nucleic acid. In certain embodiments, a portion is a defined numberof contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

“Prevent” refers to delaying or forestalling the onset or development ofa disease, disorder, or condition for a period of time from minutes toindefinitely. Prevent also means reducing risk of developing a disease,disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Protein tyrosine phosphatase 1B” or “PTP1B” (also known as PTPN1;protein tyrosine phosphatase, non-receptor type 1; PTP-1B; RKPTP) meansany nucleic acid or protein of PTP1B.

“PTP1B expression” means the level of mRNA transcribed from the geneencoding PTP1B or the level of protein translated from the mRNA. PTP1Bexpression can be determined by art known methods such as a Northern orWestern blot.

“PTP1B nucleic acid” means any nucleic acid encoding PTP1B. For example,in certain embodiments, a PTP1B nucleic acid includes a DNA sequenceencoding PTP1B, a RNA sequence transcribed from DNA encoding PTP1B(including genomic DNA comprising introns and exons), and a mRNAsequence encoding PTP1B. “PTP1B mRNA” means a mRNA encoding a PTP1Bprotein.

“Side effects” means physiological responses attributable to a treatmentother than the desired effects. In certain embodiments, side effectsinclude injection site reactions, liver function test abnormalities,renal function abnormalities, liver toxicity, renal toxicity, centralnervous system abnormalities, myopathies, and malaise. For example,increased aminotransferase levels in serum can indicate liver toxicityor liver function abnormality. For example, increased bilirubin canindicate liver toxicity or liver function abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity between an antisenseoligonucleotide and a target nucleic acid to induce a desired effect,while exhibiting minimal or no effects on non-target nucleic acids underconditions in which specific binding is desired, i.e. underphysiological conditions in the case of in vivo assays and therapeutictreatments.

“Statin” means an agent that inhibits the activity of HMG-CoA reductase.

“Subcutaneous administration” means administration just below the skin.As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “substituted sugar moiety” means a furanosyl that is nota naturally occurring sugar moiety. Substituted sugar moieties include,but are not limited to furanosyls comprising substituents at the2′-position, the 3′-position, the 5′-position and/or the 4′-position.Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside sub-units are capable of linking together and/or linking toother nucleosides to form an oligomeric compound which is capable ofhybridizing to a complementary oligomeric compound. Such structuresinclude rings comprising a different number of atoms than furanosyl(e.g., 4, 6, or 7-membered rings); replacement of the oxygen of afuranosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); orboth a change in the number of atoms and a replacement of the oxygen.Such structures may also comprise substitutions corresponding to thosedescribed for substituted sugar moieties (e.g., 6-membered carbocyclicbicyclic sugar surrogates optionally comprising additionalsubstituents). Sugar surrogates also include more complex sugarreplacements (e.g., the non-ring systems of peptide nucleic acid). Sugarsurrogates include without limitation morpholinos, cyclohexenyls andcyclohexitols.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” allrefer to a nucleic acid capable of being targeted by antisensecompounds.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of an agent thatprovides a therapeutic benefit to an individual.

“Therapeutic lifestyle change” means dietary and lifestyle changesintended to lower fat/adipose tissue mass and/or cholesterol. Suchchange can reduce the risk of developing heart disease, and may includesrecommendations for dietary intake of total daily calories, total fat,saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrate,protein, cholesterol, insoluble fiber, as well as recommendations forphysical activity.

“Triglyceride” or “TG” means a lipid or neutral fat consisting ofglycerol combined with three fatty acid molecules.

“Type 2 diabetes,” (also known as “type 2 diabetes mellitus” or“diabetes mellitus, type 2”, and formerly called “diabetes mellitus type2”, “non-insulin-dependent diabetes (NIDDM)”, “obesity relateddiabetes”, or “adult-onset diabetes”) is a metabolic disorder that isprimarily characterized by insulin resistance, relative insulindeficiency, and hyperglycemia.

“Treat” refers to administering a pharmaceutical composition to ananimal to effect an alteration or improvement of a disease, disorder, orcondition.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein, “linkage” or “linking group” means a group of atoms thatlink together two or more other groups of atoms.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “terminal internucleoside linkage” means the linkagebetween the last two nucleosides of an oligonucleotide or defined regionthereof.

As used herein, “phosphorus linking group” means a linking groupcomprising a phosphorus atom.

Phosphorus linking groups include without limitation groups having theformula:

wherein:

R_(a) and R_(d) are each, independently, O, S, CH₂, NH, or NJ₁ whereinJ₁ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

R_(b) is O or S;

R_(c) is OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, amino or substituted amino; and

J₁ is R_(b) is O or S.

Phosphorus linking groups include without limitation, phosphodiester,phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate,phosphorothioamidate, thionoalkylphosphonate, phosphotriesters,thionoalkylphosphotriester and boranophosphate.

As used herein, “internucleoside phosphorus linking group” means aphosphorus linking group that directly links two nucleosides.

As used herein, “non-internucleoside phosphorus linking group” means aphosphorus linking group that does not directly link two nucleosides. Incertain embodiments, a non-internucleoside phosphorus linking grouplinks a nucleoside to a group other than a nucleoside. In certainembodiments, a non-internucleoside phosphorus linking group links twogroups, neither of which is a nucleoside.

As used herein, “neutral linking group” means a linking group that isnot charged. Neutral linking groups include without limitationphosphotriesters, methylphosphonates, MMI (—CH₂—N(CH₃)—O—), amide-3(—CH₂—C(═O)—N(H)—), amide-4 (—CH₂—N(H)—C(═O)—), formacetal (—O—CH₂—O—),and thioformacetal (—S—CH₂—O—). Further neutral linking groups includenonionic linkages comprising siloxane (dialkylsiloxane), carboxylateester, carboxamide, sulfide, sulfonate ester and amides (See forexample: Carbohydrate Modifications in Antisense Research; Y. S. Sanghviand P. D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp.40-65)). Further neutral linking groups include nonionic linkagescomprising mixed N, O, S and CH₂ component parts.

As used herein, “internucleoside neutral linking group” means a neutrallinking group that directly links two nucleosides.

As used herein, “non-internucleoside neutral linking group” means aneutral linking group that does not directly link two nucleosides. Incertain embodiments, a non-internucleoside neutral linking group links anucleoside to a group other than a nucleoside. In certain embodiments, anon-internucleoside neutral linking group links two groups, neither ofwhich is a nucleoside.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide. Oligomeric compounds alsoinclude naturally occurring nucleic acids. In certain embodiments, anoligomeric compound comprises a backbone of one or more linked monomericsubunits where each linked monomeric subunit is directly or indirectlyattached to a heterocyclic base moiety. In certain embodiments,oligomeric compounds may also include monomeric subunits that are notlinked to a heterocyclic base moiety, thereby providing abasic sites. Incertain embodiments, the linkages joining the monomeric subunits, thesugar moieties or surrogates and the heterocyclic base moieties can beindependently modified. In certain embodiments, the linkage-sugar unit,which may or may not include a heterocyclic base, may be substitutedwith a mimetic such as the monomers in peptide nucleic acids.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” or “conjugate group” means an atom or groupof atoms bound to an oligonucleotide or oligomeric compound. In general,conjugate groups modify one or more properties of the compound to whichthey are attached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linker” or “linker” in the context of aconjugate group means a portion of a conjugate group comprising any atomor group of atoms and which covalently link (1) an oligonucleotide toanother portion of the conjugate group or (2) two or more portions ofthe conjugate group.

Conjugate groups are shown herein as radicals, providing a bond forforming covalent attachment to an oligomeric compound such as anantisense oligonucleotide. In certain embodiments, the point ofattachment on the oligomeric compound is the 3′-oxygen atom of the3′-hydroxyl group of the 3′ terminal nucleoside of the oligomericcompound. In certain embodiments the point of attachment on theoligomeric compound is the 5′-oxygen atom of the 5′-hydroxyl group ofthe 5′ terminal nucleoside of the oligomeric compound. In certainembodiments, the bond for forming attachment to the oligomeric compoundis a cleavable bond. In certain such embodiments, such cleavable bondconstitutes all or part of a cleavable moiety.

In certain embodiments, conjugate groups comprise a cleavable moiety(e.g., a cleavable bond or cleavable nucleoside) and a carbohydratecluster portion, such as a GalNAc cluster portion. Such carbohydratecluster portion comprises: a targeting moiety and, optionally, aconjugate linker. In certain embodiments, the carbohydrate clusterportion is identified by the number and identity of the ligand. Forexample, in certain embodiments, the carbohydrate cluster portioncomprises 3 GalNAc groups and is designated “GalNAc₃”. In certainembodiments, the carbohydrate cluster portion comprises 4 GalNAc groupsand is designated “GalNAc4”. Specific carbohydrate cluster portions(having specific tether, branching and conjugate linker groups) aredescribed herein and designated by Roman numeral followed by subscript“a”. Accordingly “GalNac3-1_(a)” refers to a specific carbohydratecluster portion of a conjugate group having 3 GalNac groups andspecifically identified tether, branching and linking groups. Suchcarbohydrate cluster fragment is attached to an oligomeric compound viaa cleavable moiety, such as a cleavable bond or cleavable nucleoside.

As used herein, “cleavable moiety” means a bond or group that is capableof being split under physiological conditions. In certain embodiments, acleavable moiety is cleaved inside a cell or sub-cellular compartments,such as a lysosome. In certain embodiments, a cleavable moiety iscleaved by endogenous enzymes, such as nucleases. In certainembodiments, a cleavable moiety comprises a group of atoms having one,two, three, four, or more than four cleavable bonds.

As used herein, “cleavable bond” means any chemical bond capable ofbeing split. In certain embodiments, a cleavable bond is selected fromamong: an amide, a polyamide, an ester, an ether, one or both esters ofa phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or apeptide.

As used herein, “carbohydrate cluster” means a compound having one ormore carbohydrate residues attached to a scaffold or linker group. (see,e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugatedto a Multivalent Carbohydrate Cluster for Cellular Targeting,”Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated hereinby reference in its entirety, or Rensen et al., “Design and Synthesis ofNovel N-Acetylgalactosamine-Terminated Glycolipids for Targeting ofLipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem.2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

As used herein, “modified carbohydrate” means any carbohydrate havingone or more chemical modifications relative to naturally occurringcarbohydrates.

As used herein, “carbohydrate derivative” means any compound which maybe synthesized using a carbohydrate as a starting material orintermediate.

As used herein, “carbohydrate” means a naturally occurring carbohydrate,a modified carbohydrate, or a carbohydrate derivative.

As used herein “protecting group” means any compound or protecting groupknown to those having skill in the art. Non-limiting examples ofprotecting groups may be found in “Protective Groups in OrganicChemistry”, T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley& Sons, Inc, New York, which is incorporated herein by reference in itsentirety.

As used herein, “single-stranded” means an oligomeric compound that isnot hybridized to its complement and which lacks sufficientself-complementarity to form a stable self-duplex.

As used herein, “double stranded” means a pair of oligomeric compoundsthat are hybridized to one another or a single self-complementaryoligomeric compound that forms a hairpin structure. In certainembodiments, a double-stranded oligomeric compound comprises a first anda second oligomeric compound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid. In certain embodiments, antisenseactivity includes modulation of the amount or activity of a targetnucleic acid transcript (e.g. mRNA). In certain embodiments, antisenseactivity includes modulation of the splicing of pre-mRNA.

As used herein, “RNase H based antisense compound” means an antisensecompound wherein at least some of the antisense activity of theantisense compound is attributable to hybridization of the antisensecompound to a target nucleic acid and subsequent cleavage of the targetnucleic acid by RNase H.

As used herein, “RISC based antisense compound” means an antisensecompound wherein at least some of the antisense activity of theantisense compound is attributable to the RNA Induced Silencing Complex(RISC).

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measurable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenlyation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound is intended to hybridize to result in adesired antisense activity. Antisense oligonucleotides have sufficientcomplementarity to their target nucleic acids to allow hybridizationunder physiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity. Complementary oligomeric compounds need not havenucleobase complementarity at each nucleoside. Rather, some mismatchesare tolerated. In certain embodiments, complementary oligomericcompounds or regions are complementary at 70% of the nucleobases (70%complementary). In certain embodiments, complementary oligomericcompounds or regions are 80% complementary. In certain embodiments,complementary oligomeric compounds or regions are 90% complementary. Incertain embodiments, complementary oligomeric compounds or regions are95% complementary. In certain embodiments, complementary oligomericcompounds or regions are 100% complementary.

As used herein, “mismatch” means a nucleobase of a first oligomericcompound that is not capable of pairing with a nucleobase at acorresponding position of a second oligomeric compound, when the firstand second oligomeric compound are aligned. Either or both of the firstand second oligomeric compounds may be oligonucleotides.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site.

As used herein, “fully complementary” in reference to an oligonucleotideor portion thereof means that each nucleobase of the oligonucleotide orportion thereof is capable of pairing with a nucleobase of acomplementary nucleic acid or contiguous portion thereof. Thus, a fullycomplementary region comprises no mismatches or unhybridized nucleobasesin either strand.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “chemical motif” means a pattern of chemicalmodifications in an oligonucleotide or a region thereof. Motifs may bedefined by modifications at certain nucleosides and/or at certainlinking groups of an oligonucleotide.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligonucleotide or a region thereof. The linkages ofsuch an oligonucleotide may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only nucleosides are intended to benucleoside motifs. Thus, in such instances, the linkages are notlimited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligonucleotide or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligonucleotide or region thereof. The nucleosides of such anoligonucleotide may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleosides have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “separate regions” means portions of an oligonucleotidewherein the chemical modifications or the motif of chemicalmodifications of any neighboring portions include at least onedifference to allow the separate regions to be distinguished from oneanother.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein the term “metabolic disorder” means a disease orcondition principally characterized by dysregulation of metabolism—thecomplex set of chemical reactions associated with breakdown of food toproduce energy.

As used herein, the term “cardiovascular disorder” means a disease orcondition principally characterized by impaired function of the heart orblood vessels.

As used herein the term “mono or polycyclic ring system” is meant toinclude all ring systems selected from single or polycyclic radical ringsystems wherein the rings are fused or linked and is meant to beinclusive of single and mixed ring systems individually selected fromaliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl,heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such monoand poly cyclic structures can contain rings that each have the samelevel of saturation or each, independently, have varying degrees ofsaturation including fully saturated, partially saturated or fullyunsaturated. Each ring can comprise ring atoms selected from C, N, O andS to give rise to heterocyclic rings as well as rings comprising only Cring atoms which can be present in a mixed motif such as for examplebenzimidazole wherein one ring has only carbon ring atoms and the fusedring has two nitrogen atoms. The mono or polycyclic ring system can befurther substituted with substituent groups such as for examplephthalimide which has two ═O groups attached to one of the rings. Monoor polycyclic ring systems can be attached to parent molecules usingvarious strategies such as directly through a ring atom, fused throughmultiple ring atoms, through a substituent group or through abifunctional linking moiety.

As used herein, “prodrug” means an inactive or less active form of acompound which, when administered to a subject, is metabolized to formthe active, or more active, compound (e.g., drug).

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substuent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentdisclosure have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms that differs from theatom or a group of atoms normally present in the named functional group.

In certain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino (═NR_(bb)), amido (—C(O)N—(R_(bb))(R_(cc))or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN),carbamido (—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S(O)₂R_(bb)). Whereineach R_(aa), R_(bb) and R_(cc) is, independently, H, an optionallylinked chemical functional group or a further substituent group with apreferred list including without limitation, alkyl, alkenyl, alkynyl,aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,heterocyclic and heteroarylalkyl. Selected substituents within thecompounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or poly-cyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

As used herein, “conjugate compound” means any atoms, group of atoms, orgroup of linked atoms suitable for use as a conjugate group. In certainembodiments, conjugate compounds may possess or impart one or moreproperties, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, unless otherwise indicated or modified, the term“double-stranded” refers to two separate oligomeric compounds that arehybridized to one another. Such double stranded compounds may have oneor more or non-hybridizing nucleosides at one or both ends of one orboth strands (overhangs) and/or one or more internal non-hybridizingnucleosides (mismatches) provided there is sufficient complementarity tomaintain hybridization under physiologically relevant conditions.

B. Certain Compounds

In certain embodiments, the invention provides conjugated antisensecompounds comprising antisense oligonucleotides and a conjugate.

a. Certain Antisense Oligonucleotides

In certain embodiments, the invention provides antisenseoligonucleotides. Such antisense oligonucleotides comprise linkednucleosides, each nucleoside comprising a sugar moiety and a nucleobase.The structure of such antisense oligonucleotides may be considered interms of chemical features (e.g., modifications and patterns ofmodifications) and nucleobase sequence (e.g., sequence of antisenseoligonucleotide, identity and sequence of target nucleic acid).

i. Certain Chemistry Features

In certain embodiments, antisense oligonucleotide comprise one or moremodification. In certain such embodiments, antisense oligonucleotidescomprise one or more modified nucleosides and/or modifiedinternucleoside linkages. In certain embodiments, modified nucleosidescomprise a modified sugar moiety and/or modified nucleobase.

1. Certain Sugar Moieties

In certain embodiments, compounds of the disclosure comprise one or moremodified nucleosides comprising a modified sugar moiety. Such compoundscomprising one or more sugar-modified nucleosides may have desirableproperties, such as enhanced nuclease stability or increased bindingaffinity with a target nucleic acid relative to an oligonucleotidecomprising only nucleosides comprising naturally occurring sugarmoieties. In certain embodiments, modified sugar moieties aresubstituted sugar moieties. In certain embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certainembodiments, substituted sugars comprise more than one non-bridgingsugar substituent, for example, 2′-F-5′-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′, (CH₂)₂-2′,4′-(CH₂)₃-2′, 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see,e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008);4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g., WO2009/006478,published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see,e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see,e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH₂—O—N(R)-2′, and4′-CH₂—N(R)—O-2′-, wherein each R is, independently, H, a protectinggroup, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl,or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23,2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya, et al., J. Org.Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ and analogs thereof(see, published PCT International Application WO 2008/154401, publishedon Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; U.S. Pat. Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surrogates comprise a 4′-sulfur atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a morphlino. Morpholino compounds and their use in oligomericcompounds has been reported in numerous patents and published articles(see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; andU.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As usedhere, the term “morpholino” means a sugar surrogate having the followingstructure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

For another example, in certain embodiments, a sugar surrogate comprisesa six-membered tetrahydropyran. Such tetrahydropyrans may be furthermodified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VI:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VI:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VI areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VI areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, the present disclosure provides oligonucleotidescomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingoligonucleotides possess desirable characteristics. In certainembodiments, oligonucleotides comprise one or more RNA-like nucleosides.In certain embodiments, oligonucleotides comprise one or more DNA-likenucleotides.

2. Certain Nucleobase Modifications

In certain embodiments, nucleosides of the present disclosure compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present disclosure comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

3. Certain Internucleoside Linkages

In certain embodiments, the present disclosure provides oligonucleotidescomprising linked nucleosides. In such embodiments, nucleosides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (PO), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (PS). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of theoligonucleotide. In certain embodiments, internucleoside linkages havinga chiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

4. Certain Motifs

In certain embodiments, antisense oligonucleotides comprise one or moremodified nucleoside (e.g., nucleoside comprising a modified sugar and/ormodified nucleobase) and/or one or more modified internucleosidelinkage. The pattern of such modifications on an oligonucleotide isreferred to herein as a motif. In certain embodiments, sugar,nucleobase, and linkage motifs are independent of one another.

a. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar modification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar motif, which comprises two external regionsor “wings” and a central or internal region or “gap.” The three regionsof a gapmer sugar motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar motifs of the two wings are the same as oneanother (symmetric sugar gapmer). In certain embodiments, the sugarmotifs of the 5′-wing differs from the sugar motif of the 3′-wing(asymmetric sugar gapmer).

i. Certain 5′-Wings

In certain embodiments, the 5′-wing of a gapmer consists of 1 to 8linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 1 to 7 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 1 to 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of2 to 5 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 3 to 5 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 4 or 5 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 1 to 4 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of1 to 3 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 or 2 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 2 to 4 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 2 or 3 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of3 or 4 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 nucleoside. In certain embodiments, the 5′-wing ofa gapmer consists of 2 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 3 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 4 linked nucleosides.In certain embodiments, the 5′-wing of a gapmer consists of 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of6 linked nucleosides.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmercomprises at least two bicyclic nucleosides. In certain embodiments, the5′-wing of a gapmer comprises at least three bicyclic nucleosides. Incertain embodiments, the 5′-wing of a gapmer comprises at least fourbicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 5′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 5′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 5′-wing ofa gapmer comprises at least one 2′-substituted nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one 2′-OMe nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a non-bicyclic modified nucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is a2′-substituted nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments,each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 5′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 5′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

ii. Certain 3′-Wings

In certain embodiments, the 3′-wing of a gapmer consists of 1 to 8linked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 1 to 7 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 1 to 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of2 to 5 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 3 to 5 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 4 or 5 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 1 to 4 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of1 to 3 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 or 2 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 2 to 4 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 2 or 3 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of3 or 4 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 nucleoside. In certain embodiments, the 3′-wing ofa gapmer consists of 2 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 3 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 4 linked nucleosides.In certain embodiments, the 3′-wing of a gapmer consists of 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of6 linked nucleosides.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 3′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 3′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least two non-bicyclic modified nucleosides. Incertain embodiments, the 3′-wing of a gapmer comprises at least threenon-bicyclic modified nucleosides. In certain embodiments, the 3′-wingof a gapmer comprises at least four non-bicyclic modified nucleosides.In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-substituted nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is anon-bicyclic modified nucleoside. In certain embodiments, eachnucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is a2′-MOE nucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 3′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneLNA nucleoside and at least one non-bicyclic modified nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-substituted nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside and atleast one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one LNA nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one non-bicyclic modified nucleoside, andat least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least one constrained ethyl nucleoside, at leastone non-bicyclic modified nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one LNA nucleoside, at least one non-bicyclicmodified nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-substituted nucleoside, and atleast one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one constrained ethyl nucleoside, at least one2′-substituted nucleoside, and at least one 2′-deoxynucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside, at least one 2′-substituted nucleoside, and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-MOEnucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-OMenucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.

iii. Certain Central Regions (Gaps)

In certain embodiments, the gap of a gapmer consists of 6 to 20 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6to 15 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 to 12 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 6 to 10 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 6 to 8 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6or 7 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 7 to 10 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 7 to 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 8 to 10 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 8or 9 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 linked nucleosides. In certain embodiments, the gap of agapmer consists of 7 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 8 linked nucleosides. In certain embodiments,the gap of a gapmer consists of 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 10 linked nucleosides. Incertain embodiments, the gap of a gapmer consists of 11 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 12linked nucleosides.

In certain embodiments, each nucleoside of the gap of a gapmer is a2′-deoxynucleoside. In certain embodiments, the gap comprises one ormore modified nucleosides. In certain embodiments, each nucleoside ofthe gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleosidethat is “DNA-like.” In such embodiments, “DNA-like” means that thenucleoside has similar characteristics to DNA, such that a duplexcomprising the gapmer and an RNA molecule is capable of activating RNaseH. For example, under certain conditions, 2′-(ara)-F have been shown tosupport RNase H activation, and thus is DNA-like. In certainembodiments, one or more nucleosides of the gap of a gapmer is not a2′-deoxynucleoside and is not DNA-like. In certain such embodiments, thegapmer nonetheless supports RNase H activation (e.g., by virtue of thenumber or placement of the non-DNA nucleosides).

In certain embodiments, gaps comprise a stretch of unmodified2′-deoxynucleoside interrupted by one or more modified nucleosides, thusresulting in three sub-regions (two stretches of one or more2′-deoxynucleosides and a stretch of one or more interrupting modifiednucleosides). In certain embodiments, no stretch of unmodified2′-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certainembodiments, such short stretches is achieved by using short gapregions. In certain embodiments, short stretches are achieved byinterrupting a longer gap region.

In certain embodiments, the gap comprises one or more modifiednucleosides. In certain embodiments, the gap comprises one or moremodified nucleosides selected from among cEt, FHNA, LNA, and2-thio-thymidine. In certain embodiments, the gap comprises one modifiednucleoside. In certain embodiments, the gap comprises a 5′-substitutedsugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certainembodiments, the gap comprises two modified nucleosides. In certainembodiments, the gap comprises three modified nucleosides. In certainembodiments, the gap comprises four modified nucleosides. In certainembodiments, the gap comprises two or more modified nucleosides and eachmodified nucleoside is the same. In certain embodiments, the gapcomprises two or more modified nucleosides and each modified nucleosideis different.

In certain embodiments, the gap comprises one or more modified linkages.In certain embodiments, the gap comprises one or more methyl phosphonatelinkages. In certain embodiments the gap comprises two or more modifiedlinkages. In certain embodiments, the gap comprises one or more modifiedlinkages and one or more modified nucleosides. In certain embodiments,the gap comprises one modified linkage and one modified nucleoside. Incertain embodiments, the gap comprises two modified linkages and two ormore modified nucleosides.

b. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present disclosure comprise a region ofuniformly modified internucleoside linkages. In certain suchembodiments, the oligonucleotide comprises a region that is uniformlylinked by phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide is uniformly linked by phosphorothioateinternucleoside linkages. In certain embodiments, each internucleosidelinkage of the oligonucleotide is selected from phosphodiester andphosphorothioate. In certain embodiments, each internucleoside linkageof the oligonucleotide is selected from phosphodiester andphosphorothioate and at least one internucleoside linkage isphosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 7 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least8 phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 9 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least 11 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises at least 12 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least 13phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 14 phosphorothioate internucleosidelinkages.

In certain embodiments, the oligonucleotide comprises at least one blockof at least 6 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 7 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 9 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide. In certain embodiments, the oligonucleotide comprisesless than 15 phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises less than 14 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises less than 13 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises less than 12phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises less than 11 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises lessthan 10 phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises less than 9 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises less than 8 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises less than 7phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises less than 6 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises lessthan 5 phosphorothioate internucleoside linkages.

c. Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certain suchembodiments, nucleobase modifications are arranged in a gapped motif. Incertain embodiments, nucleobase modifications are arranged in analternating motif. In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

In certain embodiments, chemical modifications to nucleobases compriseattachment of certain conjugate groups to nucleobases. In certainembodiments, each purine or each pyrimidine in an oligonucleotide may beoptionally modified to comprise a conjugate group.

d. Certain Overall Lengths

In certain embodiments, the present disclosure provides oligonucleotidesof any of a variety of ranges of lengths. In certain embodiments,oligonucleotides consist of X to Y linked nucleosides, where Xrepresents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≦Y. For example, in certainembodiments, the oligonucleotide may consist of 8 to 9, 8 to 10, 8 to11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to30 linked nucleosides. In embodiments where the number of nucleosides ofan oligonucleotide of a compound is limited, whether to a range or to aspecific number, the compound may, nonetheless further compriseadditional other substituents. For example, an oligonucleotidecomprising 8-30 nucleosides excludes oligonucleotides having 31nucleosides, but, unless otherwise indicated, such an oligonucleotidemay further comprise, for example one or more conjugate groups, terminalgroups, or other substituents.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range.

5. Certain Antisense Oligonucleotide Chemistry Motifs

In certain embodiments, the chemical structural features of antisenseoligonucleotides are characterized by their sugar motif, internucleosidelinkage motif, nucleobase modification motif and overall length. Incertain embodiments, such parameters are each independent of oneanother. Thus, each internucleoside linkage of an oligonucleotide havinga gapmer sugar motif may be modified or unmodified and may or may notfollow the gapmer modification pattern of the sugar modifications. Thus,the internucleoside linkages within the wing regions of a sugar-gapmermay be the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region. Likewise,such sugar-gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.One of skill in the art will appreciate that such motifs may be combinedto create a variety of oligonucleotides.

In certain embodiments, the selection of internucleoside linkage andnucleoside modification are not independent of one another.

i. Certain Sequences and Targets

In certain embodiments, the invention provides antisenseoligonucleotides having a sequence complementary to a target nucleicacid. Such antisense compounds are capable of hybridizing to a targetnucleic acid, resulting in at least one antisense activity. In certainembodiments, antisense compounds specifically hybridize to one or moretarget nucleic acid. In certain embodiments, a specifically hybridizingantisense compound has a nucleobase sequence comprising a region havingsufficient complementarity to a target nucleic acid to allowhybridization and result in antisense activity and insufficientcomplementarity to any non-target so as to avoid or reduce non-specifichybridization to non-target nucleic acid sequences under conditions inwhich specific hybridization is desired (e.g., under physiologicalconditions for in vivo or therapeutic uses, and under conditions inwhich assays are performed in the case of in vitro assays). In certainembodiments, oligonucleotides are selective between a target andnon-target, even though both target and non-target comprise the targetsequence. In such embodiments, selectivity may result from relativeaccessibility of the target region of one nucleic acid molecule comparedto the other.

In certain embodiments, the present disclosure provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

In certain embodiments, oligonucleotides comprise a hybridizing regionand a terminal region. In certain such embodiments, the hybridizingregion consists of 12-30 linked nucleosides and is fully complementaryto the target nucleic acid. In certain embodiments, the hybridizingregion includes one mismatch relative to the target nucleic acid. Incertain embodiments, the hybridizing region includes two mismatchesrelative to the target nucleic acid. In certain embodiments, thehybridizing region includes three mismatches relative to the targetnucleic acid. In certain embodiments, the terminal region consists of1-4 terminal nucleosides. In certain embodiments, the terminalnucleosides are at the 3′ end. In certain embodiments, one or more ofthe terminal nucleosides are not complementary to the target nucleicacid.

Antisense mechanisms include any mechanism involving the hybridizationof an oligonucleotide with target nucleic acid, wherein thehybridization results in a biological effect. In certain embodiments,such hybridization results in either target nucleic acid degradation oroccupancy with concomitant inhibition or stimulation of the cellularmachinery involving, for example, translation, transcription, orsplicing of the target nucleic acid.

One type of antisense mechanism involving degradation of target RNA isRNase H mediated antisense. RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. It is known in the art thatsingle-stranded antisense compounds which are “DNA-like” elicit RNase Hactivity in mammalian cells. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof DNA-like oligonucleotide-mediated inhibition of gene expression.

In certain embodiments, a conjugate group comprises a cleavable moiety.In certain embodiments, a conjugate group comprises one or morecleavable bond. In certain embodiments, a conjugate group comprises alinker. In certain embodiments, a linker comprises a protein bindingmoiety. In certain embodiments, a conjugate group comprises acell-targeting moiety (also referred to as a cell-targeting group). Incertain embodiments a cell-targeting moiety comprises a branching group.In certain embodiments, a cell-targeting moiety comprises one or moretethers. In certain embodiments, a cell-targeting moiety comprises acarbohydrate or carbohydrate cluster.

ii. Certain Cleavable Moieties

In certain embodiments, a cleavable moiety is a cleavable bond. Incertain embodiments, a cleavable moiety comprises a cleavable bond. Incertain embodiments, the conjugate group comprises a cleavable moiety.In certain such embodiments, the cleavable moiety attaches to theantisense oligonucleotide. In certain such embodiments, the cleavablemoiety attaches directly to the cell-targeting moiety. In certain suchembodiments, the cleavable moiety attaches to the conjugate linker. Incertain embodiments, the cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is acleavable nucleoside or nucleoside analog. In certain embodiments, thenucleoside or nucleoside analog comprises an optionally protectedheterocyclic base selected from a purine, substituted purine, pyrimidineor substituted pyrimidine. In certain embodiments, the cleavable moietyis a nucleoside comprising an optionally protected heterocyclic baseselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certainembodiments, the cleavable moiety is 2′-deoxy nucleoside that isattached to the 3′ position of the antisense oligonucleotide by aphosphodiester linkage and is attached to the linker by a phosphodiesteror phosphorothioate linkage. In certain embodiments, the cleavablemoiety is 2′-deoxy adenosine that is attached to the 3′ position of theantisense oligonucleotide by a phosphodiester linkage and is attached tothe linker by a phosphodiester or phosphorothioate linkage. In certainembodiments, the cleavable moiety is 2′-deoxy adenosine that is attachedto the 3′ position of the antisense oligonucleotide by a phosphodiesterlinkage and is attached to the linker by a phosphodiester linkage.

In certain embodiments, the cleavable moiety is attached to the 3′position of the antisense oligonucleotide. In certain embodiments, thecleavable moiety is attached to the 5′ position of the antisenseoligonucleotide. In certain embodiments, the cleavable moiety isattached to a 2′ position of the antisense oligonucleotide. In certainembodiments, the cleavable moiety is attached to the antisenseoligonucleotide by a phosphodiester linkage. In certain embodiments, thecleavable moiety is attached to the linker by either a phosphodiester ora phosphorothioate linkage. In certain embodiments, the cleavable moietyis attached to the linker by a phosphodiester linkage. In certainembodiments, the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is cleaved after thecomplex has been administered to an animal only after being internalizedby a targeted cell. Inside the cell the cleavable moiety is cleavedthereby releasing the active antisense oligonucleotide. While notwanting to be bound by theory it is believed that the cleavable moietyis cleaved by one or more nucleases within the cell. In certainembodiments, the one or more nucleases cleave the phosphodiester linkagebetween the cleavable moiety and the linker. In certain embodiments, thecleavable moiety has a structure selected from among the following:

wherein each of Bx, Bx₁, Bx₂, and Bx₃ is independently a heterocyclicbase moiety. In certain embodiments, the cleavable moiety has astructure selected from among the following:

iii. Certain Linkers

In certain embodiments, the conjugate groups comprise a linker. Incertain such embodiments, the linker is covalently bound to thecleavable moiety. In certain such embodiments, the linker is covalentlybound to the antisense oligonucleotide. In certain embodiments, thelinker is covalently bound to a cell-targeting moiety. In certainembodiments, the linker further comprises a covalent attachment to asolid support. In certain embodiments, the linker further comprises acovalent attachment to a protein binding moiety. In certain embodiments,the linker further comprises a covalent attachment to a solid supportand further comprises a covalent attachment to a protein binding moiety.In certain embodiments, the linker includes multiple positions forattachment of tethered ligands. In certain embodiments, the linkerincludes multiple positions for attachment of tethered ligands and isnot attached to a branching group. In certain embodiments, the linkerfurther comprises one or more cleavable bond. In certain embodiments,the conjugate group does not include a linker.

In certain embodiments, the linker includes at least a linear groupcomprising groups selected from alkyl, amide, disulfide, polyethyleneglycol, ether, thioether (—S—) and hydroxylamino (—O—N(H)—) groups. Incertain embodiments, the linear group comprises groups selected fromalkyl, amide and ether groups. In certain embodiments, the linear groupcomprises groups selected from alkyl and ether groups. In certainembodiments, the linear group comprises at least one phosphorus linkinggroup. In certain embodiments, the linear group comprises at least onephosphodiester group. In certain embodiments, the linear group includesat least one neutral linking group. In certain embodiments, the lineargroup is covalently attached to the cell-targeting moiety and thecleavable moiety. In certain embodiments, the linear group is covalentlyattached to the cell-targeting moiety and the antisense oligonucleotide.In certain embodiments, the linear group is covalently attached to thecell-targeting moiety, the cleavable moiety and a solid support. Incertain embodiments, the linear group is covalently attached to thecell-targeting moiety, the cleavable moiety, a solid support and aprotein binding moiety. In certain embodiments, the linear groupincludes one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalentlyattached to a scaffold group. In certain embodiments, the scaffoldincludes a branched aliphatic group comprising groups selected fromalkyl, amide, disulfide, polyethylene glycol, ether, thioether andhydroxylamino groups. In certain embodiments, the scaffold includes abranched aliphatic group comprising groups selected from alkyl, amideand ether groups. In certain embodiments, the scaffold includes at leastone mono or polycyclic ring system. In certain embodiments, the scaffoldincludes at least two mono or polycyclic ring systems. In certainembodiments, the linear group is covalently attached to the scaffoldgroup and the scaffold group is covalently attached to the cleavablemoiety and the linker. In certain embodiments, the linear group iscovalently attached to the scaffold group and the scaffold group iscovalently attached to the cleavable moiety, the linker and a solidsupport. In certain embodiments, the linear group is covalently attachedto the scaffold group and the scaffold group is covalently attached tothe cleavable moiety, the linker and a protein binding moiety. Incertain embodiments, the linear group is covalently attached to thescaffold group and the scaffold group is covalently attached to thecleavable moiety, the linker, a protein binding moiety and a solidsupport. In certain embodiments, the scaffold group includes one or morecleavable bond.

In certain embodiments, the linker includes a protein binding moiety. Incertain embodiments, the protein binding moiety is a lipid such as forexample including but not limited to cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A,vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g.,monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide, polysaccharide), an endosomolytic component, a steroid(e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid. In certain embodiments, the protein binding moietyis a C16 to C22 long chain saturated or unsaturated fatty acid,cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein each L is, independently, a phosphorus linking group or aneutral linking group; and

each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

iv. Certain Cell-Targeting Moieties

In certain embodiments, conjugate groups comprise cell-targetingmoieties. Certain such cell-targeting moieties increase cellular uptakeof antisense compounds. In certain embodiments, cell-targeting moietiescomprise a branching group, one or more tether, and one or more ligand.In certain embodiments, cell-targeting moieties comprise a branchinggroup, one or more tether, one or more ligand and one or more cleavablebond.

1. Certain Branching Groups

In certain embodiments, the conjugate groups comprise a targeting moietycomprising a branching group and at least two tethered ligands. Incertain embodiments, the branching group attaches the conjugate linker.In certain embodiments, the branching group attaches the cleavablemoiety. In certain embodiments, the branching group attaches theantisense oligonucleotide. In certain embodiments, the branching groupis covalently attached to the linker and each of the tethered ligands.In certain embodiments, the branching group comprises a branchedaliphatic group comprising groups selected from alkyl, amide, disulfide,polyethylene glycol, ether, thioether and hydroxylamino groups. Incertain embodiments, the branching group comprises groups selected fromalkyl, amide and ether groups. In certain embodiments, the branchinggroup comprises groups selected from alkyl and ether groups. In certainembodiments, the branching group comprises a mono or polycyclic ringsystem. In certain embodiments, the branching group comprises one ormore cleavable bond. In certain embodiments, the conjugate group doesnot include a branching group.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each n is, independently, from 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

-   -   wherein A₁ is O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

2. Certain Tethers

In certain embodiments, conjugate groups comprise one or more tetherscovalently attached to the branching group. In certain embodiments,conjugate groups comprise one or more tethers covalently attached to thelinking group. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, ether,thioether, disulfide, amide and polyethylene glycol groups in anycombination. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, substitutedalkyl, ether, thioether, disulfide, amide, phosphodiester andpolyethylene glycol groups in any combination. In certain embodiments,each tether is a linear aliphatic group comprising one or more groupsselected from alkyl, ether and amide groups in any combination. Incertain embodiments, each tether is a linear aliphatic group comprisingone or more groups selected from alkyl, substituted alkyl,phosphodiester, ether and amide groups in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl and phosphodiester in any combination.In certain embodiments, each tether comprises at least one phosphoruslinking group or neutral linking group.

In certain embodiments, the tether includes one or more cleavable bond.In certain embodiments, the tether is attached to the branching groupthrough either an amide or an ether group. In certain embodiments, thetether is attached to the branching group through a phosphodiestergroup. In certain embodiments, the tether is attached to the branchinggroup through a phosphorus linking group or neutral linking group. Incertain embodiments, the tether is attached to the branching groupthrough an ether group. In certain embodiments, the tether is attachedto the ligand through either an amide or an ether group. In certainembodiments, the tether is attached to the ligand through an ethergroup. In certain embodiments, the tether is attached to the ligandthrough either an amide or an ether group. In certain embodiments, thetether is attached to the ligand through an ether group.

In certain embodiments, each tether comprises from about 8 to about 20atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether group comprises from about 10 to about18 atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether group comprises about 13 atoms in chainlength.

In certain embodiments, a tether has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:

-   -   wherein L is either a phosphorus linking group or a neutral        linking group;    -   Z₁ is C(═O)O—R₂;    -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;    -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein Z₂ is H or CH₃; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

-   -   In certain embodiments, a tether comprises a phosphorus linking        group. In certain embodiments, a tether does not comprise any        amide bonds. In certain embodiments, a tether comprises a        phosphorus linking group and does not comprise any amide bonds.

3. Certain Ligands

In certain embodiments, the present disclosure provides ligands whereineach ligand is covalently attached to a tether. In certain embodiments,each ligand is selected to have an affinity for at least one type ofreceptor on a target cell. In certain embodiments, ligands are selectedthat have an affinity for at least one type of receptor on the surfaceof a mammalian liver cell. In certain embodiments, ligands are selectedthat have an affinity for the hepatic asialoglycoprotein receptor(ASGP-R). In certain embodiments, each ligand is a carbohydrate. Incertain embodiments, each ligand is, independently selected fromgalactose, N-acetyl galactoseamine, mannose, glucose, glucosamone andfucose. In certain embodiments, each ligand is N-acetyl galactoseamine(GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6ligands. In certain embodiments, the targeting moiety comprises 3ligands. In certain embodiments, the targeting moiety comprises 3N-acetyl galactoseamine ligands.

In certain embodiments, the ligand is a carbohydrate, carbohydratederivative, modified carbohydrate, multivalent carbohydrate cluster,polysaccharide, modified polysaccharide, or polysaccharide derivative.In certain embodiments, the ligand is an amino sugar or a thio sugar.For example, amino sugars may be selected from any number of compoundsknown in the art, for example glucosamine, sialic acid,α-D-galactosamine, N-Acetylgalactosamine,2-acetamido-2-deoxy-D-galactopyranose (GalNAc),2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramicacid), 2-Deoxy-2-methylamino-L-glucopyranose,4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, andN-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selectedfrom the group consisting of 5-Thio-β-D-glucopyranose, Methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-Thio-β-D-galactopyranose, and ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, “GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in theliterature as N-acetyl galactosamine. In certain embodiments, “N-acetylgalactosamine” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. Incertain embodiments, “GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments,“GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both the(3-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, boththe β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably.Accordingly, in structures in which one form is depicted, thesestructures are intended to include the other form as well. For example,where the structure for an α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure isintended to include the other form as well. In certain embodiments, Incertain preferred embodiments, the β-form2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

In certain embodiments one or more ligand has a structure selected fromamong:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected fromamong:

In certain embodiments one or more ligand has a structure selected fromamong:

In certain embodiments one or more ligand has a structure selected fromamong:

i. Certain Conjugates

In certain embodiments, conjugate groups comprise the structuralfeatures above. In certain such embodiments, conjugate groups have thefollowing structure:

wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

wherein each n is, independently, from 1 to 20;

Z is H or a linked solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain embodiments, conjugates do not comprise a pyrrolidine.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of six to elevenconsecutively bonded atoms.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of ten consecutivelybonded atoms.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to elevenconsecutively bonded atoms and wherein the tether comprises exactly oneamide bond.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising anether, a ketone, an amide, an ester, a carbamate, an amine, apiperidine, a phosphate, a phosphodiester, a phosphorothioate, atriazole, a pyrrolidine, a disulfide, or a thioether.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl group, or a group comprising exactly one ether orexactly two ethers, an amide, an amine, a piperidine, a phosphate, aphosphodiester, or a phosphorothioate.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl group.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, and 12.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms, and wherein X does not comprise an ethergroup.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of eightconsecutively bonded atoms, and wherein X does not comprise an ethergroup.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms, and wherein the tether comprises exactly oneamide bond, and wherein X does not comprise an ether group.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms and wherein the tether consists of an amidebond and a substituted or unsubstituted C₂-C₁₁ alkyl group.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl,alkenyl, or alkynyl group, or a group comprising an ether, a ketone, anamide, an ester, a carbamate, an amine, a piperidine, a phosphate, aphosphodiester, a phosphorothioate, a triazole, a pyrrolidine, adisulfide, or a thioether.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkylgroup, or a group comprising an ether, an amine, a piperidine, aphosphate, a phosphodiester, or a phosphorothioate.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkylgroup.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein n is 4, 5, 6, 7, or 8.

In certain embodiments, conjugates do not comprise a pyrrolidine.

b. Certain Conjugated Antisense Compounds

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside. In certain embodiments, a conjugated antisense compound hasthe following structure:

-A-B-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

-A-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least onecleavable bond.

In certain such embodiments, the branching group comprises at least onecleavable bond.

In certain embodiments each tether comprises at least one cleavablebond.

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

-A-B-CE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside. In certain embodiments, a conjugated antisense compound hasthe following structure:

-A-CE-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

-A-B-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

-A-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least onecleavable bond.

In certain embodiments each tether comprises at least one cleavablebond.

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

Representative United States patents, United States patent applicationpublications, and international patent application publications thatteach the preparation of certain of the above noted conjugates,conjugated antisense compounds, tethers, linkers, branching groups,ligands, cleavable moieties as well as other modifications includewithout limitation, U.S. Pat. No. 5,994,517, U.S. Pat. No. 6,300,319,U.S. Pat. No. 6,660,720, U.S. Pat. No. 6,906,182, U.S. Pat. No.7,262,177, U.S. Pat. No. 7,491,805, U.S. Pat. No. 8,106,022, U.S. Pat.No. 7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO2012/037254, each of which is incorporated by reference herein in itsentirety.

Representative publications that teach the preparation of certain of theabove noted conjugates, conjugated antisense compounds, tethers,linkers, branching groups, ligands, cleavable moieties as well as othermodifications include without limitation, BIESSEN et al., “TheCholesterol Derivative of a Triantennary Galactoside with High Affinityfor the Hepatic Asialoglycoprotein Receptor: a Potent CholesterolLowering Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al.,“Synthesis of Cluster Galactosides with High Affinity for the HepaticAsialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE etal., “New and more efficient multivalent glyco-ligands forasialoglycoprotein receptor of mammalian hepatocytes” Bioorganic &Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., “Determinationof the Upper Size Limit for Uptake and Processing of Ligands by theAsialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J.Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., “Design andSynthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids forTargeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J.Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesisof Novel Amphiphilic Dendritic Galactosides for Selective Targeting ofLiposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem.(1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis oflysine-based cluster galactosides with high affinity for theAsialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each ofwhich is incorporated by reference herein in its entirety.

In certain embodiments, conjugated antisense compounds comprise an RNaseH based oligonucleotide (such as a gapmer) or a splice modulatingoligonucleotide (such as a fully modified oligonucleotide) and anyconjugate group comprising at least one, two, or three GalNAc groups. Incertain embodiments a conjugated antisense compound comprises anyconjugate group found in any of the following references: Lee, CarbohydrRes, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257,939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee etal., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987,4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676;Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al.,Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38,3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al.,Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276,37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlindet al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med ChemLett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007,15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Leeet al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., AnalytBiochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012,51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852;Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J MedChem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol,2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464;Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J OrgChem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792;Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., MethodsEnzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14,18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan,Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al.,Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013,21, 5275-5281; International applications WO1998/013381; WO2011/038356;WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254;WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947;WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046;WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013;WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709;WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406;WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat.Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. PatentApplication Publications US2011/0097264; US2011/0097265; US2013/0004427;US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730;US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814;US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393;US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075;US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938;US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968;US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; eachof which is incorporated by reference in its entirety.

C. Certain Uses and Features

In certain embodiments, conjugated antisense compounds exhibit potenttarget RNA reduction in vivo. In certain embodiments, unconjugatedantisense compounds accumulate in the kidney. In certain embodiments,conjugated antisense compounds accumulate in the liver. In certainembodiments, conjugated antisense compounds are well tolerated. Suchproperties render conjugated antisense compounds particularly useful forinhibition of many target RNAs, including, but not limited to thoseinvolved in metabolic, cardiovascular and other diseases, disorders orconditions. Thus, provided herein are methods of treating such diseases,disorders or conditions by contacting liver tissues with the conjugatedantisense compounds targeted to RNAs associated with such diseases,disorders or conditions. Thus, also provided are methods forameliorating any of a variety of metabolic, cardiovascular and otherdiseases, disorders or conditions with the conjugated antisensecompounds of the present invention.

In certain embodiments, conjugated antisense compounds are more potentthan unconjugated counterpart at a particular tissue concentration.Without wishing to be bound by any theory or mechanism, in certainembodiments, the conjugate may allow the conjugated antisense compoundto enter the cell more efficiently or to enter the cell moreproductively. For example, in certain embodiments conjugated antisensecompounds may exhibit greater target reduction as compared to itsunconjugated counterpart wherein both the conjugated antisense compoundand its unconjugated counterpart are present in the tissue at the sameconcentrations. For example, in certain embodiments conjugated antisensecompounds may exhibit greater target reduction as compared to itsunconjugated counterpart wherein both the conjugated antisense compoundand its unconjugated counterpart are present in the liver at the sameconcentrations.

Productive and non-productive uptake of oligonucleotides has beeddiscussed previously (See e.g. Geary, R. S., E. Wancewicz, et al.(2009). “Effect of Dose and Plasma Concentration on Liver Uptake andPharmacologic Activity of a 2′-Methoxyethyl Modified Chimeric AntisenseOligonucleotide Targeting PTEN.” Biochem. Pharmacol. 78(3): 284-91; &Koller, E., T. M. Vincent, et al. (2011). “Mechanisms of single-strandedphosphorothioate modified antisense oligonucleotide accumulation inhepatocytes.” Nucleic Acids Res. 39(11): 4795-807). Conjugate groupsdescribed herein may improve productive uptake.

In certain embodiments, the conjugate groups described herein mayfurther improve potency by increasing the affinity of the conjugatedantisense compound for a particular type of cell or tissue. In certainembodiments, the conjugate groups described herein may further improvepotency by increasing recognition of the conjugated antisense compoundby one or more cell-surface receptors. In certain embodiments, theconjugate groups described herein may further improve potency byfacilitating endocytosis of the conjugated antisense compound.

In certain embodiments, the cleavable moiety may further improve potencyby allowing the conjugate to be cleaved from the antisenseoligonucleotide after the conjugated antisense compound has entered thecell. Accordingly, in certain embodiments, conjugated antisensecompounds can be administered at doses lower than would be necessary forunconjugated antisense oligonucleotides.

Phosphorothioate linkages have been incorporated into antisenseoligonucleotides previously. Such phosphorothioate linkages areresistant to nucleases and so improve stability of the oligonucleotide.Further, phosphorothioate linkages also bind certain proteins, whichresults in accumulation of antisense oligonucleotide in the liver.Oligonucleotides with fewer phosphorothioate linkages accumulate less inthe liver and more in the kidney (see, for example, Geary, R.,“Pharmacokinetic Properties of 2′-O-(2-Methoxyethyl)-ModifiedOligonucleotide Analogs in Rats,” Journal of Pharmacology andExperimental Therapeutics, Vol. 296, No. 3, 890-897; & PharmacologicalProperties of 2′-O-Methoxyethyl Modified Oligonucleotides in Antisense aDrug Technology, Chapter 10, Crooke, S. T., ed., 2008) In certainembodiments, oligonucleotides with fewer phosphorothioateinternculeoside linkages and more phosphodiester internucleosidelinkages accumulate less in the liver and more in the kidney. Whentreating diseases in the liver, this is undesirable for several reasons(1) less drug is getting to the site of desired action (liver); (2) drugis escaping into the urine; and (3) the kidney is exposed to relativelyhigh concentration of drug which can result in toxicities in the kidney.Thus, for liver diseases, phosphorothioate linkages provide importantbenefits.

In certain embodiments, however, administration of oligonucleotidesuniformly linked by phosphoro-thioate internucleoside linkages inducesone or more proinflammatory reactions. (see for example: J Lab Clin Med.1996 September; 128(3):329-38. “Amplification of antibody production byphosphorothioate oligodeoxynucleotides”. Branda et al.; and see also forexample: Toxicologic Properties in Antisense a Drug Technology, Chapter12, pages 342-351, Crooke, S. T., ed., 2008). In certain embodiments,administration of oligonucleotides wherein most of the internucleosidelinkages comprise phosphorothioate internucleoside linkages induces oneor more proinflammatory reactions.

In certain embodiments, the degree of proinflammatory effect may dependon several variables (e.g. backbone modification, off-target effects,nucleobase modifications, and/or nucleoside modifications) see forexample: Toxicologic Properties in Antisense a Drug Technology, Chapter12, pages 342-351, Crooke, S. T., ed., 2008). In certain embodiments,the degree of proinflammatory effect may be mitigated by adjusting oneor more variables. For example the degree of proinflammatory effect of agiven oligonucleotide may be mitigated by replacing any number ofphosphorothioate internucleoside linkages with phosphodiesterinternucleoside linkages and thereby reducing the total number ofphosphorothioate internucleoside linkages.

In certain embodiments, it would be desirable to reduce the number ofphosphorothioate linkages, if doing so could be done without losingstability and without shifting the distribution from liver to kidney.For example, in certain embodiments, the number of phosphorothioatelinkages may be reduced by replacing phosphorothioate linkages withphosphodiester linkages. In such an embodiment, the antisense compoundhaving fewer phosphorothioate linkages and more phosphodiester linkagesmay induce less proinflammatory reactions or no proinflammatoryreaction. Although the antisense compound having fewer phosphoro-thioatelinkages and more phosphodiester linkages may induce fewerproinflammatory reactions, the antisense compound having fewerphosphorothioate linkages and more phosphodiester linkages may notaccumulate in the liver and may be less efficacious at the same orsimilar dose as compared to an antisense compound having morephosphorothioate linkages. In certain embodiments, it is thereforedesirable to design an antisense compound that has a plurality ofphosphodiester bonds and a plurality of phosphorothioate bonds but whichalso possesses stability and good distribution to the liver.

In certain embodiments, conjugated antisense compounds accumulate morein the liver and less in the kidney than unconjugated counterparts, evenwhen some of the phosporothioate linkages are replaced with lessproinflammatory phosphodiester internucleoside linkages. In certainembodiments, conjugated antisense compounds accumulate more in the liverand are not excreted as much in the urine compared to its unconjugatedcounterparts, even when some of the phosporothioate linkages arereplaced with less proinflammatory phosphodiester internucleosidelinkages. In certain embodiments, the use of a conjugate allows one todesign more potent and better tolerated antisense drugs. Indeed, incertain embodiments, conjugated antisense compounds have largertherapeutic indexes than unconjugated counterparts. This allows theconjugated antisense compound to be administered at a higher absolutedose, because there is less risk of proinflammatory response and lessrisk of kidney toxicity. This higher dose, allows one to dose lessfrequently, since the clearance (metabolism) is expected to be similar.Further, because the compound is more potent, as described above, onecan allow the concentration to go lower before the next dose withoutlosing therapeutic activity, allowing for even longer periods betweendosing.

In certain embodiments, the inclusion of some phosphorothioate linkagesremains desirable. For example, the terminal linkages are vulnerable toexonucleases and so in certain embodiments, those linkages arephosphorothioate or other modified linkage. Internucleoside linkageslinking two deoxynucleosides are vulnerable to endonucleases and so incertain embodiments those linkages are phosphorothioate or othermodified linkage. Internucleoside linkages between a modified nucleosideand a deoxynucleoside where the deoxynucleoside is on the 5′ side of thelinkage deoxynucleosides are vulnerable to endonucleases and so incertain embodiments those linkages are phosphorothioate or othermodified linkage. Internucleoside linkages between two modifiednucleosides of certain types and between a deoxynucleoside and amodified nucleoside of certain type where the modified nucleoside is atthe 5′ side of the linkage are sufficiently resistant to nucleasedigestion, that the linkage can be phosphodiester.

In certain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 16 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 15 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 14 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 13 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 12 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 11 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 10 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 9 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 8 phosphorthioate linkages.

In certain embodiments, antisense compounds comprising one or moreconjugate group described herein has increased activity and/or potencyand/or tolerability compared to a parent antisense compound lacking suchone or more conjugate group. Accordingly, in certain embodiments,attachment of such conjugate groups to an oligonucleotide is desirable.Such conjugate groups may be attached at the 5′-, and/or 3′-end of anoligonucleotide. In certain instances, attachment at the 5′-end issynthetically desirable. Typically, oligonucleotides are synthesized byattachment of the 3′ terminal nucleoside to a solid support andsequential coupling of nucleosides from 3′ to 5′ using techniques thatare well known in the art. Accordingly if a conjugate group is desiredat the 3′-terminus, one may (1) attach the conjugate group to the3′-terminal nucleoside and attach that conjugated nucleoside to thesolid support for subsequent preparation of the oligonucleotide or (2)attach the conjugate group to the 3′-terminal nucleoside of a completedoligonucleotide after synthesis. Neither of these approaches is veryefficient and thus both are costly. In particular, attachment of theconjugated nucleoside to the solid support, while demonstrated in theExamples herein, is an inefficient process. In certain embodiments,attaching a conjugate group to the 5′-terminal nucleoside issynthetically easier than attachment at the 3′-end. One may attach anon-conjugated 3′ terminal nucleoside to the solid support and preparethe oligonucleotide using standard and well characterized reactions. Onethen needs only to attach a 5′nucleoside having a conjugate group at thefinal coupling step. In certain embodiments, this is more efficient thanattaching a conjugated nucleoside directly to the solid support as istypically done to prepare a 3′-conjugated oligonucleotide. The Examplesherein demonstrate attachment at the 5′-end. In addition, certainconjugate groups have synthetic advantages. For Example, certainconjugate groups comprising phosphorus linkage groups are syntheticallysimpler and more efficiently prepared than other conjugate groups,including conjugate groups reported previously (e.g., WO/2012/037254).

In certain embodiments, conjugated antisense compounds are administeredto a subject. In such embodiments, antisense compounds comprising one ormore conjugate group described herein has increased activity and/orpotency and/or tolerability compared to a parent antisense compoundlacking such one or more conjugate group. Without being bound bymechanism, it is believed that the conjugate group helps withdistribution, delivery, and/or uptake into a target cell or tissue. Incertain embodiments, once inside the target cell or tissue, it isdesirable that all or part of the conjugate group to be cleaved torelease the active oligonucleotide. In certain embodiments, it is notnecessary that the entire conjugate group be cleaved from theoligonucleotide. For example, in Example 20 a conjugated oligonucleotidewas administered to mice and a number of different chemical species,each comprising a different portion of the conjugate group remaining onthe oligonucleotide, were detected (Table 10a). This conjugatedantisense compound demonstrated good potency (Table 10). Thus, incertain embodiments, such metabolite profile of multiple partialcleavage of the conjugate group does not interfere withactivity/potency. Nevertheless, in certain embodiments it is desirablethat a prodrug (conjugated oligonucleotide) yield a single activecompound. In certain instances, if multiple forms of the active compoundare found, it may be necessary to determine relative amounts andactivities for each one. In certain embodiments where regulatory reviewis required (e.g., USFDA or counterpart) it is desirable to have asingle (or predominantly single) active species. In certain suchembodiments, it is desirable that such single active species be theantisense oligonucleotide lacking any portion of the conjugate group. Incertain embodiments, conjugate groups at the 5′-end are more likely toresult in complete metabolism of the conjugate group. Without beingbound by mechanism it may be that endogenous enzymes responsible formetabolism at the 5′ end (e.g., 5′ nucleases) are more active/efficientthan the 3′ counterparts.

In certain embodiments, the specific conjugate groups are more amenableto metabolism to a single active species. In certain embodiments,certain conjugate groups are more amenable to metabolism to theoligonucleotide.

D. Antisense

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. In such embodiments, the oligomeric compound iscomplementary to a target nucleic acid. In certain embodiments, a targetnucleic acid is an RNA. In certain embodiments, a target nucleic acid isa non-coding RNA. In certain embodiments, a target nucleic acid encodesa protein. In certain embodiments, a target nucleic acid is selectedfrom a mRNA, a pre-mRNA, a microRNA, a non-coding RNA, including smallnon-coding RNA, and a promoter-directed RNA. In certain embodiments,oligomeric compounds are at least partially complementary to more thanone target nucleic acid. For example, oligomeric compounds of thepresent invention may be microRNA mimics, which typically bind tomultiple targets.

In certain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 70% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence at least 80%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 90% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence at least 95%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 98% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence that is 100%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds are at least 70%, 80%, 90%,95%, 98%, or 100% complementary to the nucleobase sequence of a targetnucleic acid over the entire length of the antisense compound.

Antisense mechanisms include any mechanism involving the hybridizationof an oligomeric compound with target nucleic acid, wherein thehybridization results in a biological effect. In certain embodiments,such hybridization results in either target nucleic acid degradation oroccupancy with concomitant inhibition or stimulation of the cellularmachinery involving, for example, translation, transcription, orpolyadenylation of the target nucleic acid or of a nucleic acid withwhich the target nucleic acid may otherwise interact.

One type of antisense mechanism involving degradation of target RNA isRNase H mediated antisense. RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. It is known in the art thatsingle-stranded antisense compounds which are “DNA-like” elicit RNase Hactivity in mammalian cells. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof DNA-like oligonucleotide-mediated inhibition of gene expression.

Antisense mechanisms also include, without limitation RNAi mechanisms,which utilize the RISC pathway. Such RNAi mechanisms include, withoutlimitation siRNA, ssRNA and microRNA mechanisms. Such mechanisms includecreation of a microRNA mimic and/or an anti-microRNA.

Antisense mechanisms also include, without limitation, mechanisms thathybridize or mimic non-coding RNA other than microRNA or mRNA. Suchnon-coding RNA includes, but is not limited to promoter-directed RNA andshort and long RNA that effects transcription or translation of one ormore nucleic acids.

In certain embodiments, oligonucleotides comprising conjugates describedherein are RNAi compounds. In certain embodiments, oligomericoligonucleotides comprising conjugates described herein are ssRNAcompounds. In certain embodiments, oligonucleotides comprisingconjugates described herein are paired with a second oligomeric compoundto form an siRNA. In certain such embodiments, the second oligomericcompound also comprises a conjugate. In certain embodiments, the secondoligomeric compound is any modified or unmodified nucleic acid. Incertain embodiments, the oligonucleotides comprising conjugatesdescribed herein is the antisense strand in an siRNA compound. Incertain embodiments, the oligonucleotides comprising conjugatesdescribed herein is the sense strand in an siRNA compound. Inembodiments in which the conjugated oligomeric compound isdouble-stranded siRnA, the conjugate may be on the sense strand, theantisense strand or both the sense strand and the antisense strand.

D. Target Nucleic Acids, Regions and Segments

In certain embodiments, conjugated antisense compounds target anynucleic acid. In certain embodiments, the target nucleic acid encodes atarget protein that is clinically relevant. In such embodiments,modulation of the target nucleic acid results in clinical benefit.Certain target nucleic acids include, but are not limited to, the targetnucleic acids illustrated in Table 1.

TABLE 1 Certain Human Target Nucleic Acids Target GENBANK ® AccessionNumber SEQ ID NO PTP1B NM_002827.2 1 NT_011362.9 truncated fromnucleotides 2 14178000 to 14256000 FGFR4 NM_002011.3 3 NT_023133.11truncated from nucleosides 4 21323018 to 21335213 AB209631.1 5NM_022963.2 6 GCCR the complement of GENBANK Accession 7 No.NT_029289.10 truncated from nucleotides 3818000 to 3980000 GCGRNM_000160.3 8 NW_926918.1 truncated from nucleotides 9 16865000 to16885000 Factor VII NT_027140.6 truncated from nucleotides 10 1255000 to1273000 NM_019616.2 11 DB184141.1 12 NW_001104507.1 truncated from 13nucleotides 691000 to 706000 Factor XI NM_000128.3 14 NT_022792.17,truncated from 19598000 15 to 19624000 NM_028066.1 16 NW_001118167.1 17

The targeting process usually includes determination of at least onetarget region, segment, or site within the target nucleic acid for theantisense interaction to occur such that the desired effect will result.

In certain embodiments, a target region is a structurally defined regionof the nucleic acid. For example, in certain such embodiments, a targetregion may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region or target segment.

In certain embodiments, a target segment is at least about an8-nucleobase portion of a target region to which a conjugated antisensecompound is targeted. Target segments can include DNA or RNA sequencesthat comprise at least 8 consecutive nucleobases from the 5′-terminus ofone of the target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA comprises about 8 to about 30 nucleobases). Targetsegments are also represented by DNA or RNA sequences that comprise atleast 8 consecutive nucleobases from the 3′-terminus of one of thetarget segments (the remaining nucleobases being a consecutive stretchof the same DNA or RNA beginning immediately downstream of the3′-terminus of the target segment and continuing until the DNA or RNAcomprises about 8 to about 30 nucleobases). Target segments can also berepresented by DNA or RNA sequences that comprise at least 8 consecutivenucleobases from an internal portion of the sequence of a targetsegment, and may extend in either or both directions until theconjugated antisense compound comprises about 8 to about 30 nucleobases.

In certain embodiments, antisense compounds targeted to the nucleicacids listed in Table 1 can be modified as described herein. In certainembodiments, the antisense compounds can have a modified sugar moiety,an unmodified sugar moiety or a mixture of modified and unmodified sugarmoieties as described herein. In certain embodiments, the antisensecompounds can have a modified internucleoside linkage, an unmodifiedinternucleoside linkage or a mixture of modified and unmodifiedinternucleoside linkages as described herein. In certain embodiments,the antisense compounds can have a modified nucleobase, an unmodifiednucleobase or a mixture of modified and unmodified nucleobases asdescribed herein. In certain embodiments, the antisense compounds canhave a motif as described herein.

In certain embodiments, antisense compounds targeted to the nucleicacids listed in Table 1 can be conjugated as described herein.

1. Protein Tyrosine Phosphatase 1B (PTP1B)

Protein tyrosine phosphatase 1B (PTP1B) is a member of a family of PTPs(Barford, et al., Science 1994. 263: 1397-1404) and is a cytosolicenzyme (Neel and Tonks, Curr. Opin. Cell Biol. 1997. 9: 193-204). PTP1Bis expressed ubiquitously including tissues that are key regulators ofinsulin metabolism such as liver, muscle and fat (Goldstein, Receptor1993. 3: 1-15), where it is the main PTP enzyme.

PTP1B is considered to be a negative regulator of insulin signalingPTP1B interacts with and dephosphorylates the insulin receptor, thusattenuating and potentially terminating the insulin signalingtransduction (Goldstein et al., J. Biol. Chem. 2000. 275: 4383-4389).The physiological role of PTP1B in insulin signaling has beendemonstrated in knockout mice models. Mice lacking the PTP1B gene wereprotected against insulin resistance and obesity (Elchebly et al.,Science 1999. 283: 1544-1548). PTP1B-deficient mice had low adiposity,increased basal metabolic rate as well as total energy expenditure andwere protected from diet-induced obesity. Insulin-stimulated glucoseuptake was elevated in skeletal muscle, whereas adipose tissue wasunaffected providing evidence that increased insulin sensitivity inPTP1B-deficient mice was tissue-specific (Klaman et al., Mol. Cell.Biol. 2000. 20: 5479-5489). These mice were phenotypically normal andwere also resistant to diet-induced obesity, insulin resistance and hadsignificantly lower triglyceride levels on a high-fat diet. Therefore,inhibition of PTP1B in patients suffering from Type II diabetes,metabolic syndrome, diabetic dyslipidemia, or related metabolic diseaseswould be beneficial.

Antisense inhibition of PTP1B provides a unique advantage overtraditional small molecule inhibitors in that antisense inhibitors donot rely on competitive binding of the compound to the protein andinhibit activity directly by reducing the expression of PTP1B. Antisensetechnology is emerging as an effective means for reducing the expressionof certain gene products and may therefore prove to be uniquely usefulin a number of therapeutic, diagnostic, and research applications forthe modulation of PTP1B.

There is a currently a lack of acceptable options for treating metabolicdisorders. It is therefore an object herein to provide compounds andmethods for the treatment of such diseases and disorder.

All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated-by-reference forthe portions of the document discussed herein, as well as in theirentirety.

Certain Conjugated Antisense Compounds Targeted to a PTP1B Nucleic Acid

In certain embodiments, conjugated antisense compounds are targeted to aPTP1B nucleic acid having the sequence of GENBANK® Accession No.NM_002827.2, incorporated herein as SEQ ID NO: 1 or GENBANK AccessionNo. NT_011362.9 truncated from nucleotides 14178000 to Ser. No.14/256,000, incorporated herein as SEQ ID NO: 2. In certain suchembodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 isat least 90%, at least 95%, or 100% complementary to SEQ ID NOs: 1and/or 2.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 54. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO:54.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 55. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO:55.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 56. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO:56.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 57. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO:57.

TABLE 2 Antisense Compounds targeted to PTP1B SEQ ID NO: 1 Target SEQStart ID ISIS No Site Sequence (5′-3′) Motif NO 142082 3291AAATGGTTTATTCCATGGCC 5-10-5 MOE 54 404173 3290 AATGGTTTATTCCATGGCCA5-10-5 MOE 55 409826 3287 GGTTTATTCCATGGCCATTG 5-10-5 MOE 56 446431 3292AATGGTTTATTCCATGGC 4-10-4 MOE 57

In certain embodiments, a compound comprises or consists of ISIS 142082and a conjugate group. ISIS 142082 is a modified oligonucleotide havingthe formula: Aes Aes Aes Tes Ges Gds Tds Tds Tds Ads Tds Tds mCds mCdsAds Tes Ges Ges mCes mCe, wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 404173and a conjugate group. ISIS 404173 is a modified oligonucleotide havingthe formula: Aes Aes Tes Ges Ges Tds Tds Tds Ads Tds Tds mCds mCds AdsTds Ges Ges mCes mCes Ae, wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 409826and a conjugate group. ISIS 409826 is a modified oligonucleotide havingthe formula: Ges Ges Tes Tes Tes Ads Tds Tds mCds mCds Ads Tds Gds GdsmCds mCes Aes Tes Tes Ge, wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 446431and a conjugate group. ISIS 446431 is a modified oligonucleotide havingthe formula: Aes Aes Tes Ges Gds Tds Tds Tds Ads Tds Tds mCds mCds AdsTes Ges Ges mCe, wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises an antisenseoligonucleotide disclosed in U.S. Pat. No. 7,563,884 and WO 2007/131237,which is incorporated by reference in its entirety herein, and aconjugate group described herein. In certain embodiments, a compoundcomprises an antisense oligonucleotide having a nucleobase sequence ofany of SEQ ID NOs 17-96 and 244-389 disclosed in U.S. Pat. No. 7,563,884and a conjugate group described herein. In certain embodiments, acompound comprises an antisense oligonucleotide having a nucleobasesequence of any of SEQ ID NOs 886-1552 of SEQ ID Nos WO 2007/131237 anda conjugate group described herein. In certain embodiments, a compoundcomprises an antisense oligonucleotide disclosed in U.S. Pat. No.7,563,884 and WO 2007/131237, which is incorporated by reference in itsentirety herein, and a conjugate group described herein. In certainembodiments, a compound comprises an antisense oligonucleotide having anucleobase sequence of any of SEQ ID NOs disclosed in U.S. Pat. No.7,563,884 and WO 2007/131237 and a conjugate group described herein. Thenucleobase sequences of all of the aforementioned referenced SEQ ID NOsare incorporated by reference herein.

PTP1B Therapeutic Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more conjugatedpharmaceutical compositions as described herein. In certain embodiments,the individual has metabolic related disease.

As shown in the examples below, conjugated compounds targeted to PTP1B,as described herein, have been shown to reduce the severity ofphysiological symptoms of metabolic related diseases, includingmetabolic syndrome, diabetes mellitus, insulin resistance, diabeticdyslipidemia, hypertriglyceridemia, obesity and weight gain. In certainof the experiments, the conjugated compounds reduced blood glucoselevels, e.g., the animals continued to experience symptoms, but thesymptoms were less severe compared to untreated animals. In other of theexperiments, however, the conjugated compounds appear to reduce thesymptoms of diabetes; e.g., animals treated for a longer period of timeexperienced less severe symptoms than those administered the compoundsfor a shorter period of time. In other of the experiments, however, theconjugated compounds appear to inhibit weight gain; e.g., animalstreated for a longer period of time experienced less severe symptomsthan those administered the compounds for a shorter period of time. Inother of the experiments, however, the conjugated compounds appear toinhibit hypertriglyceridemia; e.g., animals treated for a longer periodof time experienced less severe symptoms than those administered thecompounds for a shorter period of time. The ability of the conjugatedcompounds exemplified below to restore function therefore demonstratesthat symptoms of the disease may be reversed by treatment with acompound as described herein.

Diabetes mellitus is characterized by numerous physical andphysiological symptoms. Any symptom known to one of skill in the art tobe associated with Type 2 diabetes can be ameliorated or otherwisemodulated as set forth above in the methods described above. In certainembodiments, the symptom is a physical symptom selected from the groupconsisting of increased glucose levels, increased weight gain, frequenturination, unusual thirst, extreme hunger, extreme fatigue, blurredvision, frequent infections, tingling or numbness at the extremities,dry and itchy skin, weight loss, slow-healing sores, and swollen gums.

In certain embodiments, the symptom is a physiological symptom selectedfrom the group consisting of increased insulin resistance, increasedglucose levels, increased fat mass, decreased metabolic rate, decreasedglucose clearance, decreased glucose tolerance, decreased insulinsensitivity, decreased hepatic insulin sensitivity, increased adiposetissue size and weight, increased body fat, and increased body weight.

Liu and Chernoff have shown that PTP1B binds to and serves as asubstrate for the epidermal growth factor receptor (EGFR) (Liu andChernoff, Biochem. J., 1997, 327, 139-145). Furthermore, in A431 humanepidermoid carcinoma cells, PT1B was found to be inactivated by thepresence of H₂O₂ generated by the addition of EGF. These studiesindicate that PTP1B can be negatively regulated by the oxidation stateof the cell, which is often deregulated during tumorigenesis (Lee etal., J. Biol. Chem., 1998, 273, 15366-15372).

Overexpression of PTP1B has been demonstrated in malignant ovariancancers and this correlation was accompanied by a concomitant increasein the expression of the associated growth factor receptor (Wiener etal., Am. J. Obstet. Gynecol., 1994, 170, 1177-1183).

PTP1B has been shown to suppress transformation in NIH3T3 cells inducedby the neu oncogene (Brown-Shimer et al., Cancer Res., 1992, 52,478-482), as well as in rat 3Y1 fibroblasts induced by v-srk, v-src, andv-ras (Liu et al., Mol. Cell. Biol., 1998, 18, 250-259) and rat-1fibroblasts induced by bcr-abl (LaMontagne et al., Proc. Natl. Acad.Sci. U S. A., 1998, 95, 14094-14099). It has also been shown that PTP1Bpromotes differentiation of K562 cells, a chronic myelogenous leukemiacell line, in a similar manner as does an inhibitor of the bcr-abloncoprotein. These studies describe the possible role of PTP1B incontrolling the pathogenesis of chronic myeloid leukemia (LaMontagne etal., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 14094-14099).

Accordingly, provided herein are methods for ameliorating a symptomassociated with hyperproliferative disorders in a subject in needthereof. In certain embodiments, the hyperproliferative disorder iscancer. In certain embodiments, provided herein are methods forameliorating a symptom associated with cancer. In certain embodiments,provided is a method for reducing the rate of onset of a symptomassociated with hyperproliferative disorders. In certain embodiments,provided is a method for reducing the rate of onset of a symptomassociated with cancer. In certain embodiments, provided is a method forreducing the severity of a symptom associated with hyperproliferativedisorders. In certain embodiments, provided is a method for reducing theseverity of a symptom associated with cancer. In such embodiments, themethods comprise administering to an individual in need thereof atherapeutically effective amount of a compound targeted to a PTP1Bnucleic acid.

In certain embodiments, provided are methods of treating an individualcomprising administering one or more conjugated pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas metabolic related disease.

In certain embodiments, administration of a conjugated antisensecompound targeted to a PTP1B nucleic acid results in reduction of PTP1Bexpression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues.

In certain embodiments, pharmaceutical compositions comprising aconjugated antisense compound targeted to PTP1B are used for thepreparation of a medicament for treating a patient suffering orsusceptible to metabolic related disease.

In certain embodiments, the methods described herein includeadministering a compound comprising a conjugate group and a modifiedoligonucleotide having a contiguous nucleobases portion as describedherein of a sequence recited in SEQ ID NOs: 54-57.

It will be understood that any of the compounds described herein can beused in the aforementioned methods and uses. For example, in certainembodiments a conjugated antisense compound targeted to a PTP1B nucleicacid in the aforementioned methods and uses can include, but is notlimited to, a conjugated antisense compound targeted to SEQ ID NO: 1comprising an at least 8 consecutive nucleobase sequence of any of SEQID NOs: 54-57; a conjugated antisense compound targeted to SEQ ID NO: 1comprising a nucleobase sequence of any of SEQ ID NOs: 54-57; a compoundcomprising or consisting of ISIS 142082, ISIS 404173, ISIS 409826, orISIS 446431 and a conjugate group; a compound comprising an antisenseoligonucleotide disclosed in U.S. Pat. No. 7,563,884 and WO 2007/131237,which is incorporated by reference in its entirety herein, and aconjugate group; a compound comprising an antisense oligonucleotidehaving a nucleobase sequence of any of SEQ ID NOs 17-96 and 244-389disclosed in U.S. Pat. No. 7,563,884 and a conjugate group describedherein; or a compound comprising an antisense oligonucleotide having anucleobase sequence of any of—SEQ ID NOs 886-1552 disclosed in WO2007/131237 and a conjugate group described herein.

2. FGFR4

Obesity is considered a long-term metabolic disease. There are severalserious medical sequelae related to obesity. There are over 1 billionoverweight individuals worldwide with 100 million clinically obese. Theincreasing health care costs of treating obesity related diseases in theUS alone are estimated at over $100 billion annually. Current methodsfor treating obesity include behavioral modification, diet, surgery(gastroplasty), administering pharmaceutical agents that block appetitestimulating signals or absorption of nutrients (fat), and administeringagents that increase thermogenesis or fat metabolism. Some of thesemethods have disadvantages in that they rely on patient resolve, areinvasive, or have unwanted side effects. An understanding of themechanisms by which obesity is regulated may provide importanttherapeutic information.

Obesity is frequently associated with insulin resistance and togetherconstitutes risk factors for later development of type 2 diabetes andcardiovascular diseases. Insulin resistance occurs well beforedevelopment of type 2 diabetes, and insulin is overproduced tocompensate for the insulin resistance and to maintain normal glucoselevels. Type 2 diabetes ensues, as the pancreas can no longer produceenough insulin to maintain normal glucose levels. Early stages of type 2diabetes are associated with elevated levels of insulin but as thedisease progresses the pancreas may fail to produce insulin, resultingin increased blood glucose levels. Diabetes is a significant risk factorfor both heart disease and stroke and is the leading cause of blindnessand end-stage renal failure.

Diabetes is a disorder characterized by hyperglycemia due to deficientinsulin action that may result from reduced insulin production orinsulin resistance or both. Diabetes mellitus is a polygenic disorderaffecting a significant portion of the people in the world. It isdivided into two types. In type I diabetes, or insulin-dependentdiabetes mellitus (IDDM), patients produce little or no insulin, thehormone that regulates glucose utilization. In type 2 diabetes, ornoninsulin-dependent diabetes mellitus (NIDDM), patients often haveplasma insulin levels that are the same compared to nondiabetic humans;however, these patients have developed a resistance to the insulinstimulating effect of glucose and lipid metabolism in the maininsulin-sensitive tissues, i.e., muscle, liver and adipose tissues, andthe plasma insulin levels are insufficient to overcome the pronouncedinsulin resistance. Additionally, glucotoxicity, which results fromlong-term hyperglycemia, induces tissue-dependent insulin resistance(Nawano et al., Am. J. Physiol. Endocrinol. Metab., 278, E535-543)exacerbating the disease. Type 2 diabetes accounts for over 90% of alldiabetes cases. It is a metabolic disorder characterized byhyperglycemia leading to secondary complications such as neuropathy,nephropathy, retinopathy, hypertriglyceridemia, obesity, and othercardiovascular diseases generally referred to as metabolic syndrome.

Metabolic syndrome is a combination of medical disorders that increaseone's risk for cardiovascular disease and diabetes. The symptoms,including high blood pressure, high triglycerides, decreased HDL andobesity, tend to appear together in some individuals. Metabolic syndromeis known under various other names, such as (metabolic) syndrome X,insulin resistance syndrome or Reaven's syndrome.

Diabetes and obesity (sometimes now collectively referred to as“diabesity”) are interrelated in that obesity is known to exacerbate thepathology of diabetes and greater than 60% of diabetics are obese. Mosthuman obesity is associated with insulin resistance and leptinresistance. In fact, it has been suggested that obesity may have an evengreater impact on insulin action than diabetes itself (Sindelka et al.,Physiol Res., 51, 85-91). Additionally, several compounds on the marketfor the treatment of diabetes are known to induce weight gain, a veryundesirable side effect to the treatment of this disease. Therefore, acompound that has the potential to treat both diabetes and obesity wouldprovide a significant improvement over current treatments.

Fibroblast growth factor receptor 4 (also known as FGF receptor-4, TKF;tyrosine kinase related to fibroblast growth factor receptor;hydroxyaryl-protein kinase; tyrosylprotein kinase; Fgfr4; FGFR-4; FGFR4;CD334, FGFR4_HUMAN and JTK2) has high affinity for the acidic and/orbasic fibroblast growth factors. (Armstrong et al., Genes ChromosomesCancer, 4, 94-98).

Although FGFRs generally have been shown to have wide distributionthroughout the body, to date, FGFR4 has only been found in a fewtissues. Among a wide variety of cells and tissues tested, includinghuman lymphocytes and macrophages, FGFR4 was found to be expressed inthe lung and in some tumors of lung origin as well as in malignanciesnot derived from lung tissues. (Holtrich et al., Proc. Nat. Acad. Sci.,88, 10411-10415). FGFR4 has also been found to be expressed in the liverand in adipose tissues. (Patel et al., JCEM, 90(2), 1226-1232). FGFR4has also been found to be expressed in certain carcinoma cell lines.(Bange et al., Cancer Res., 62, 840-847).

Additionally, FGFR4 has been shown to play a role in systemic lipid andglucose homeostasis. FGFR4-deficient mice on a normal diet exhibitedfeatures of metabolic syndrome that include increase mass of insulinresistance, in addition to hypercholesterolemia. FGFR4 deficiency wasshown to alleviate high-fat diet-induced fatty liver in a certain obesemouse model, which is also a correlate of metabolic syndrome.Restoration of FGFR4, specifically in hepatocytes of FGFR4 deficientmice, decrease plasma lipid level and restored the high fat diet-inducedfatty liver but failed to restore glucose tolerance and sensitivity toinsulin. (Huang et al., Diabetes, 56, 2501-2510).

Antisense inhibition of FGFR4 provides a unique advantage overtraditional small molecule inhibitors in that antisense inhibitors donot rely on competitive binding of the compound to the protein andinhibit activity directly by reducing the expression of FGFR4. Arepresentative United States patent that teaches FGFR4 antisenseinhibitors includes US. Pat. Publication No. US2010/0292140, of which isherein incorporated by reference in its entirety. Antisense technologyis emerging as an effective means for reducing the expression of certaingene products and may therefore prove to be uniquely useful in a numberof therapeutic, diagnostic, and research applications for the modulationof FGFR4.

There is a currently a lack of acceptable options for treating metabolicdisorders. It is therefore an object herein to provide compounds andmethods for the treatment of such diseases and disorder. This inventionrelates to the discovery of novel, highly potent inhibitors of FGFR4gene expression.

All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated-by-reference forthe portions of the document discussed herein, as well as in theirentirety.

Certain Conjugated Antisense Compounds Targeted to a FGFR4 Nucleic Acid

In certain embodiments, conjugated antisense compounds are targeted to aFGFR4 nucleic acid having the sequence GENBANK Accession No. NM_002011.3(incorporated herein as SEQ ID NO: 3), GENBANK Accession No:NT_023133.11 truncated from nucleosides 21323018 to 21335213(incorporated herein as SEQ ID NO: 4); and GENBANK Accession No.AB209631.1 (incorporated herein as SEQ ID NO: 5); and GENBANK AccessionNo NM_022963.2 (incorporated herein as SEQ ID NO: 6). In certain suchembodiments, a conjugated antisense compound targeted to SEQ ID NOs: 3-6is at least 90%, at least 95%, or 100% complementary to SEQ ID NOs: 3-6.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of anyone of SEQ ID NOs: 58-65. In certain embodiments, a conjugated antisensecompound targeted to SEQ ID NO: 3 comprises a nucleobase sequence of anyone of SEQ ID NO: 12-19.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 58. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 3 comprises a nucleobase sequence of SEQ ID NO:59.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 59. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 3 comprises a nucleobase sequence of SEQ ID NO:59.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 60. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 3 comprises a nucleobase sequence of SEQ ID NO:60.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 61. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 3 comprises a nucleobase sequence of SEQ ID NO:61.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 62. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 3 comprises a nucleobase sequence of SEQ ID NO:62.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 63. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 3 comprises a nucleobase sequence of SEQ ID NO:63.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 64. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 3 comprises a nucleobase sequence of SEQ ID NO:64.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 3 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 65. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 3 comprises a nucleobase sequence of SEQ ID NO:65.

TABLE 3 Antisense Compounds targeted to FGFR4 SEQ ID NO: 3 Target SEQStart ID ISIS No Site Sequence (5′-3′) Motif NO 299005 192GGCACACTCAGCAGGACCCC 5-10-5 MOE 58 463588 191 GCACACTCAGCAGGACCCCC5-10-5 MOE 59 463589 193 AGGCACACTCAGCAGGACCC 5-10-5 MOE 60 463690 369GCCAGGCGACTGCCCTCCTT 5-10-5 MOE 61 463691 370 TGCCAGGCGACTGCCCTCCT5-10-5 MOE 62 463835 788 CGCTCTCCATCACGAGACTC 5-10-5 MOE 63 463837 790CACGCTCTCCATCACGAGAC 5-10-5 MOE 64 464225 2954 CTTCCAGCTTCTCTGGGCTC5-10-5 MOE 65

In certain embodiments, a compound comprises or consists of ISIS 299005and a conjugate group. ISIS 299005 is a modified oligonucleotide havingthe formula: Ges Ges mCes Aes mCes Ads mCds Tds mCds Ads Gds mCds AdsGds Gds Aes mCes mCes mCes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 463588and a conjugate group. ISIS 463588 is a modified oligonucleotide havingthe formula: Ges mCes Aes mCes Aes mCds Tds mCds Ads Gds mCds Ads GdsGds Ads mCes mCes mCes mCes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 463589and a conjugate group. ISIS 463589 is a modified oligonucleotide havingthe formula: Aes Ges Ges mCes Aes mCds Ads mCds Tds mCds Ads Gds mCdsAds Gds Ges Aes mCes mCes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 463690and a conjugate group. ISIS 463690 is a modified oligonucleotide havingthe formula: Ges mCes mCes Aes Ges Gds mCds Gds Ads mCds Tds Gds mCdsmCds mCds Tes mCes mCes Tes Te, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 463691and a conjugate group. ISIS 463691 is a modified oligonucleotide havingthe formula: Tes Ges mCes mCes Aes Gds Gds mCds Gds Ads mCds Tds GdsmCds mCds mCes Tes mCes mCes Te, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 463835and a conjugate group. ISIS 463835 is a modified oligonucleotide havingthe formula: mCes Ges mCes Tes mCes Tds mCds mCds Ads Tds mCds Ads mCdsGds Ads Ges Aes mCes Tes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 463837and a conjugate group. ISIS 463837 is a modified oligonucleotide havingthe formula: mCes Aes mCes Ges mCes Tds mCds Tds mCds mCds Ads Tds mCdsAds mCds Ges Aes Ges Aes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 464225and a conjugate group. ISIS 464225 is a modified oligonucleotide havingthe formula: mCes Tes Tes mCes mCes Ads Gds mCds Tds Tds mCds Tds mCdsTds Gds Ges Ges mCes Tes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises an antisenseoligonucleotide disclosed in WO 2009/046141, which are incorporated byreference in their entireties herein, and a conjugate group. In certainembodiments, a compound comprises an antisense oligonucleotide having anucleobase sequence of any of SEQ ID NOs 21-24, 28, 29, 36, 38, 39, 43,48, 51, 54-56, 58-60, 64-66, 92-166 disclosed in WO 2009/046141 and aconjugate group described herein. In certain embodiments, a compoundcomprises an antisense oligonucleotide having a nucleobase sequencecomplementary to a preferred target segment of any of SEQ ID NOs 21-24,28, 29, 36, 38, 39, 43, 48, 51, 54-56, 58-60, 64-66, 92-166 disclosed inWO 2009/046141 and a conjugate group described herein. The nucleobasesequences of all of the aforementioned referenced SEQ ID NOs areincorporated by reference herein.

FGFR4 Therapeutic Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas a metabolic disease.

As shown in the examples below, conjugated compounds targeted to FGFR4,as described herein, have been shown to reduce the severity ofphysiological symptoms of a metabolic disease, including obesity oradiposity, metabolic syndrome, diabetes mellitus, insulin resistance,diabetic dyslipidemia, and hypertriglyceridemia. In certain of theexperiments, the conjugated compounds reduced body weight, e.g., theanimals continued to experience symptoms, but the symptoms were lesssevere compared to untreated animals. In certain of the experiments, theconjugated compounds reduced body fat, e.g., the animals continued toexperience symptoms, but the symptoms were less severe compared tountreated animals. In certain of the experiments, the conjugatedcompounds reduced adipose tissue, e.g., the animals continued toexperience symptoms, but the symptoms were less severe compared tountreated animals. In other of the experiments, however, the conjugatedcompounds appear to reduce the symptoms of obesity; e.g., animalstreated for a longer period of time experienced less severe symptomsthan those administered the compounds for a shorter period of time. Inother of the experiments, however, the conjugated compounds appear toreduce the symptoms of diabetes; e.g., animals treated for a longerperiod of time experienced less severe symptoms than those administeredthe compounds for a shorter period of time. In other of the experiments,however, the conjugated compounds appear to inhibit weight gain; e.g.,animals treated for a longer period of time experienced less severesymptoms than those administered the compounds for a shorter period oftime. In other of the experiments, however, the conjugated compoundsappear to reduce glucose levels; e.g., animals treated for a longerperiod of time experienced less severe symptoms than those administeredthe compounds for a shorter period of time. In other of the experiments,however, the conjugated compounds appear to increase fatty acidoxidation; e.g., animals treated for a longer period of time experiencedless severe symptoms than those administered the compounds for a shorterperiod of time. The ability of the conjugated compounds exemplifiedbelow to restore function therefore demonstrates that symptoms of thedisease may be reversed by treatment with a compound as describedherein.

Obesity is characterized by numerous physical and physiologicalsymptoms. Any symptom known to one of skill in the art to be associatedwith obesity can be ameliorated or otherwise modulated as set forthabove in the methods described above. In certain embodiments, thesymptom is a physical symptom selected from the group consisting ofincreased adipose tissue mass or weight, increased weight gain,increased fat pad weight, imbalance with caloric intake and energyexpenditure, increase in body fat, increase in body mass, having a bodymass index (BMI) of 30 or higher, increase in body frame, increasedsweating, sleep apnea, difficulty in sleeping, inability to cope withsudden physical activity, lethargy, back and joint problems, increase inbreathlessness, increase in breast region adiposity, increase in abdomensize or fat, extreme hunger, or extreme fatigue.

In certain embodiments, the symptom is a physiological symptom selectedfrom the group consisting of high blood pressure, hypertension, highcholesterol levels, type 2 diabetes, stroke, cardiac insufficiency,heart disease, coronary artery obstruction, breast cancer in women,gastro-oesophageal reflux disease, hip and knee arthrosis, and reducedlife expectancy.

In certain embodiments, the physical symptom is excess body weight. Incertain embodiments, the symptom is excess fat mass. In certainembodiments, the symptom is a body mass index of 30 or higher. Incertain embodiments, the symptom is breathlessness. In certainembodiments, the symptom is increased sweating. In certain embodiments,the symptom is sleep apnea. In certain embodiments, the symptom isdifficulty in sleeping. In certain embodiments, the symptom is inabilityto cope with sudden physical activity. In certain embodiments, thesymptom is lethargy. In certain embodiments, the symptom is back andjoint problems.

In certain embodiments, the physiological symptom is high bloodpressure. In certain embodiments, the symptom is hypertension. Incertain embodiments, the symptom is high cholesterol levels. In certainembodiments, the symptom is type 2 diabetes. In certain embodiments, thesymptom is stroke. In certain embodiments, the symptom is cardiacinsufficiency. In certain embodiments, the symptom is heart disease. Incertain embodiments, the symptom is coronary artery obstruction. Incertain embodiments, the symptom is breast cancer in women. In certainembodiments, the symptom is gastro-oesophageal reflux disease. Incertain embodiments, the symptom is hip and knee arthrosis. In certainembodiments, the symptom is reduced life expectancy.

Diabetes mellitus is characterized by numerous physical andphysiological symptoms. Any symptom known to one of skill in the art tobe associated with Type 2 diabetes can be ameliorated or otherwisemodulated as set forth above in the methods described above. In certainembodiments, the symptom is a physical symptom selected from the groupconsisting of increased glucose levels, increased weight gain, frequenturination, unusual thirst, extreme hunger, extreme fatigue, blurredvision, frequent infections, tingling or numbness at the extremities,dry and itchy skin, weight loss, slow-healing sores, and swollen gums.

In certain embodiments, the symptom is a physiological symptom selectedfrom the group consisting of increased insulin resistance, increasedglucose levels, increased fat mass, decreased metabolic rate, decreasedglucose clearance, decreased glucose tolerance, decreased insulinsensitivity, decreased hepatic insulin sensitivity, increased adiposetissue size and weight, increased body fat, and increased body weight.

In certain embodiments, the physical symptom is increased weight gain.In certain embodiments, the symptom is frequent urination. In certainembodiments, the symptom is unusual thirst. In certain embodiments, thesymptom is extreme hunger. In certain embodiments, the symptom isextreme fatigue. In certain embodiments, the symptom is blurred vision.In certain embodiments, the symptom is frequent infections. In certainembodiments, the symptom is tingling or numbness at the extremities. Incertain embodiments, the symptom is dry and itchy skin. In certainembodiments, the symptom is weight loss. In certain embodiments, thesymptom is slow-healing sores. In certain embodiments, the symptom isswollen gums. In certain embodiments, the symptom is increased insulinresistance. In certain embodiments, the symptom is increased fat mass.In certain embodiments, the symptom is decreased metabolic rate. Incertain embodiments, the symptom is decreased glucose clearance. Incertain embodiments, the symptom is decreased glucose tolerance. Incertain embodiments, the symptom is decreased insulin sensitivity. Incertain embodiments, the symptom is decreased hepatic insulinsensitivity. In certain embodiments, the symptom is increased adiposetissue size and weight. In certain embodiments, the symptom is increasedbody fat. In certain embodiments, the symptom is increased body weight.

In certain embodiments, provided are methods of treating an individualcomprising administering one or more pharmaceutical compositions asdescribed herein. In certain embodiments, the individual has metabolicrelated disease.

In certain embodiments, administration of a conjugated antisensecompound targeted to a FGFR4 nucleic acid results in reduction of FGFR4expression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues.

In certain embodiments, pharmaceutical compositions comprising aconjugated antisense compound targeted to FGFR4 are used for thepreparation of a medicament for treating a patient suffering orsusceptible to a metabolic disease.

In certain embodiments, the methods described herein includeadministering a conjugated compound comprising a modifiedoligonucleotide having a contiguous nucleobases portion as describedherein of a sequence recited in SEQ ID NO: 58-65.

It will be understood that any of the compounds described herein can beused in the aforementioned methods and uses. For example, in certainembodiments a conjugated antisense compound targeted to a FGFR4 nucleicacid in the aforementioned methods and uses can include, but is notlimited to, a conjugated antisense compound targeted to SEQ ID NO: 3comprising an at least 8 consecutive nucleobase sequence of any one ofSEQ ID NOs: 58-65; a conjugated antisense compound targeted to SEQ IDNO: 3 comprising a nucleobase sequence of any one of SEQ ID NO: 58-65; acompound comprising or consisting of ISIS 299005, ISIS 463588, ISIS463589, ISIS 463690, ISIS 463691, ISIS 463835, ISIS 463837, or ISIS464225 and a conjugate group; a compound comprising an antisenseoligonucleotide disclosed in WO 2009/046141, which are incorporated byreference in their entireties herein, and a conjugate group; a compoundcomprising an antisense oligonucleotide having a nucleobase sequence ofany of SEQ ID NOs SEQ ID NOs 21-24, 28, 29, 36, 38, 39, 43, 48, 51,54-56, 58-60, 64-66, 92-166 disclosed in WO 2009/046141 and a conjugategroup described herein; or a compound comprising an antisenseoligonucleotide having a nucleobase sequence complementary to apreferred target segment of any of SEQ ID NOs 21-24, 28, 29, 36, 38, 39,43, 48, 51, 54-56, 58-60, 64-66, 92-166 disclosed in WO 2009/046141 anda conjugate group described herein. The nucleobase sequences of all ofthe aforementioned referenced SEQ ID NOs are incorporated by referenceherein.

3. GCCR

Diabetes is a chronic metabolic disorder characterized by impairedinsulin secretion and/or action. In type 2 diabetes (T2DM), insulinresistance leads to an inability of insulin to control the activity ofgluconeogenic enzymes, and many subjects also exhibit inappropriatelevels of circulating glucagon (GC) in the fasting and postprandialstate. Glucagon is secreted from the α-cells of the pancreatic isletsand regulates glucose homeostasis through modulation of hepatic glucoseproduction (Quesada et al., J. Endocrinol. 2008. 199: 5-19).

Glucagon exerts its action on target tissues via the activation ofglucocorticoid receptor (GCCR). The glucocorticoid receptor is a 62 kDaprotein that is a member of the class B G-protein coupled family ofreceptors (Brubaker et al., Recept. Channels. 2002. 8: 179-88). GCCRactivation leads to signal transduction by G proteins (G_(s)α andG_(q)), whereby G_(s)α activates adenylate cyclase, which causes cAMPproduction, resulting in an increase in levels of protein kinase A. GCCRsignaling in the liver results in increased hepatic glucose productionby induction of glycogenolysis and gluconeogenesis along with inhibitionof glycogenesis (Jiang and Zhang. Am. J. Physiol. Endocrinol. Metab.2003. 284: E671-E678). GCCR is also expressed in extrahepatic tissues,which includes heart, intestinal smooth muscle, kidney, brain, andadipose tissue (Hansen et al., Peptides. 1995. 16: 1163-1166).

Development of GCCR inhibitors have been hampered by the unfavorableside effects associated with systemic GCCR inhibition, includingactivation of the hypothalamic-pituitary adrenal (HPA) axis. Inhibitionof GCCR activity in the brain can lead to an increase in circulatingadrenocorticotropic hormone due to feedback regulation and a consequentincrease in secretion of adrenal steroids (Philibert et al., Front.Horm. Res. 1991. 19: 1-17). This, in turn, can produce a myriad ofnegative chronic steroid-related side-effects. Other studies havedemonstrated that specific inactivation of GCCR resulted in hypoglycemiaupon prolonged fasting (Opherk et al., Mol. Endocronol. 2004. 18:1346-1353).

It has previously been demonstrated in pre-clinical models thatadministration of GCCR antisense oligonucleotides results intissue-specific accumulation and reduced GCCR expression in liver andadipose tissue (PCT Pub. No. WO2005/071080; PCT Pub. No. WO2007/035759)without affecting GCCR mRNA levels in the CNS or adrenal glands. Thus,antisense inhibition of GCCR mRNA expression has be shown to improvehyperglycemia and hyperlipidemia without activating the HPA axis. Thepresent invention provides compositions and methods for modulating GCCRexpression. Antisense compounds for modulating expression of GCCR aredisclosed in the aforementioned published patent applications. However,there remains a need for additional improved compounds. The compoundsand treatment methods described herein provide significant advantagesover the treatments options currently available for GCCR relateddisorders. All documents, or portions of documents, cited in thisapplication, including, but not limited to, patents, patentapplications, articles, books, and treatises, are hereby expresslyincorporated-by-reference for the portions of the document discussedherein, as well as in their entirety.

Certain Conjugated Antisense Compounds Targeted to a GCCR Nucleic Acid

In certain embodiments, conjugated antisense compounds are targeted to aGCCR nucleic acid having the sequence the complement of GENBANKAccession No. NT_029289.10 truncated from nucleotides 3818000 to U.S.Pat. No. 3,980,000 (incorporated herein as SEQ ID NO: 7). In certainsuch embodiments, a conjugated antisense compound targeted to SEQ ID NO:7 is at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 7.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of anyone of SEQ ID NOs: 66-77. In certain embodiments, a conjugated antisensecompound targeted to SEQ ID NO: 7 comprises a nucleobase sequence of anyone of SEQ ID NO: 66-77.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 66. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:66.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 67. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:67.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 68. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:68.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 69. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:69.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 70. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:70.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 71. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:71.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 72. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:72.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 73. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:73.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 74. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:74.In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 75. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:75.In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 76. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:76.In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 7 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 77. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 7 comprises a nucleobase sequence of SEQ ID NO:77.

TABLE 4a Antisense Compounds targeted to GCCR SEQ ID NO: 7 Target SEQStart ID ISIS No Site Sequence (5′-3′) Motif NO 420470 57825GGTAGAAATATAGTTGTTCC 5-10-5 MOE 66 420476 59956 TTCATGTGTCTGCATCATGT5-10-5 MOE 67 426115 65940 GCAGCCATGGTGATCAGGAG 5-10-5 MOE 68 42613063677 GCATCCAGCGAGCACCAAAG 5-10-5 MOE 69 426168 76224GTCTGGATTACAGCATAAAC 5-10-5 MOE 70 426172 76229 CCTTGGTCTGGATTACAGCA5-10-5 MOE 71 426183 65938 AGCCATGGTGATCAGGAGGC 3-14-3 MOE 72 42624676225 GGTCTGGATTACAGCATAAA 3-14-3 MOE 73 426261 65938AGCCATGGTGATCAGGAGGC 2-13-5 MOE 74 426262 65939 CAGCCATGGTGATCAGGAGG2-13-5 MOE 75 426267 95513 GTGCTTGTCCAGGATGATGC 2-13-5 MOE 76 42632576229 CCTTGGTCTGGATTACAGCA 2-13-5 MOE 77

In certain embodiments, a compound comprises or consists of ISIS 420470and a conjugate group. ISIS 420470 is a modified oligonucleotide havingthe formula: Ges Ges Tes Aes Ges Ads Ads Ads Tds Ads Tds Ads Gds Tds TdsGes Tes Tes mCes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 420476and a conjugate group. ISIS 420476 is a modified oligonucleotide havingthe formula: Tes Tes mCes Aes Tes Gds Tds Gds Tds mCds Tds Gds mCds AdsTds mCes Aes Tes Ges Te, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426115and a conjugate group. ISIS 426115 is a modified oligonucleotide havingthe formula: Ges mCes Aes Ges mCes mCds Ads Tds Gds Gds Tds Gds Ads TdsmCds Aes Ges Ges Aes Ge, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426130and a conjugate group. ISIS 426130 is a modified oligonucleotide havingthe formula: Ges mCes Aes Tes mCes mCds Ads Gds mCds Gds Ads Gds mCdsAds mCds mCes Aes Aes Aes Ge, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426168and a conjugate group. ISIS 426168 is a modified oligonucleotide havingthe formula: Ges Tes mCes Tes Ges Gds Ads Tds Tds Ads mCds Ads Gds mCdsAds Tes Aes Aes Aes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426172and a conjugate group. ISIS 426172 is a modified oligonucleotide havingthe formula: mCes mCes Tes Tes Ges Gds Tds mCds Tds Gds Gds Ads Tds TdsAds mCes Aes Ges mCes Ae, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426183and a conjugate group. ISIS 426183 is a modified oligonucleotide havingthe formula: Aes Ges mCes mCds Ads Tds Gds Gds Tds Gds Ads Tds mCds AdsGds Gds Ads Ges Ges mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426246and a conjugate group. ISIS 426246 is a modified oligonucleotide havingthe formula: Ges Ges Tes mCds Tds Gds Gds Ads Tds Tds Ads mCds Ads GdsmCds Ads Tds Aes Aes Ae, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426261and a conjugate group. ISIS 426261 is a modified oligonucleotide havingthe formula: Aes Ges mCds mCds Ads Tds Gds Gds Tds Gds Ads Tds mCds AdsGds Ges Aes Ges Ges mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426262and a conjugate group. ISIS 426262 is a modified oligonucleotide havingthe formula: mCes Aes Gds mCds mCds Ads Tds Gds Gds Tds Gds Ads Tds mCdsAds Ges Ges Aes Ges Ge, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426267and a conjugate group. ISIS 426267 is a modified oligonucleotide havingthe formula: Ges Tes Gds mCds Tds Tds Gds Tds mCds mCds Ads Gds Gds AdsTds Ges Aes Tes Ges mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 426325and a conjugate group. ISIS 426325 is a modified oligonucleotide havingthe formula: mCes mCes Tds Tds Gds Gds Tds mCds Tds Gds Gds Ads Tds TdsAds mCes Aes Ges mCes Ae, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises an antisenseoligonucleotide disclosed in WO 2005/071080, WO 2007/035759, or WO2007/136988, which are incorporated by reference in their entiretiesherein, and a conjugate group. In certain embodiments, a compoundcomprises an antisense oligonucleotide having a nucleobase sequence ofany of SEQ ID NOs 30-216, and 306-310 disclosed in WO 2005/071080 and aconjugate group described herein. In certain embodiments, a compoundcomprises an antisense oligonucleotide having a nucleobase sequence ofany of SEQ ID NOs 26-113 disclosed in WO 2007/035759 and a conjugategroup described herein. In certain embodiments, a compound comprises anantisense oligonucleotide having a nucleobase sequence of any of SEQ IDNOs 413-485 disclosed in WO 2007/136988 and a conjugate group describedherein. In certain embodiments, a compound comprises an antisenseoligonucleotide having a nucleobase sequence complementary to apreferred target segment of any of SEQ ID NOs 30-216, and 306-310disclosed in WO 2005/071080, 26-113 disclosed in WO 2007/035759, and413-485 disclosed in WO 2007/136988, and a conjugate group describedherein. The nucleobase sequences of all of the aforementioned referencedSEQ ID NOs are incorporated by reference herein.

GCCR Therapeutic Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas metabolic related disease.

As shown in the examples below, conjugated compounds targeted to GCCR,as described herein, have been shown to reduce the severity ofphysiological symptoms of metabolic related diseases, includingmetabolic syndrome, diabetes mellitus, insulin resistance, diabeticdyslipidemia, hypertriglyceridemia, obesity and weight gain e.g., theanimals continued to experience symptoms, but the symptoms were lesssevere compared to untreated animals. In certain experiments, theconjugated compounds reduced blood glucose levels. In other experiments,the conjugated compounds reduce the symptoms of diabetes. In otherexperiments, the conjugated compounds inhibit weight gain. In otherexperiments, the conjugated compounds inhibit hypertriglyceridemia. Incertain embodiments, the conjugated compounds restore function thereforedemonstratingreversal of disease by treatment with a compound asdescribed herein. In certain embodiments, animals treated for a longerperiod of time experience less severe symptoms than those administeredthe compounds for a shorter period of time.

Diabetes mellitus is characterized by numerous physical andphysiological signs and/or symptoms. Any symptom known to one of skillin the art to be associated with Type 2 diabetes can be ameliorated orotherwise modulated as set forth above in the methods described above.In certain embodiments, the sign or symptom is a physical symptom suchas increased glucose levels, increased weight gain, frequent urination,unusual thirst, extreme hunger, extreme fatigue, blurred vision,frequent infections, tingling or numbness at the extremities, dry anditchy skin, weight loss, slow-healing sores, and swollen gums. Incertain embodiments, the sign or symptom is a physiological symptom suchas increased insulin resistance, increased glucose levels, increased fatmass, decreased metabolic rate, decreased glucose clearance, decreasedglucose tolerance, decreased insulin sensitivity, decreased hepaticinsulin sensitivity, increased adipose tissue size and weight, increasedbody fat, and increased body weight.

In certain embodiments, the physical sign or symptom is increasedglucose levels. In certain embodiments, the sign or symptom is weightgain. In certain embodiments, the sign or symptom is frequent urination.In certain embodiments, the sign or symptom is unusual thirst. Incertain embodiments, the sign or symptom is extreme hunger. In certainembodiments, the sign or symptom is extreme fatigue. In certainembodiments, the sign or symptom is blurred vision. In certainembodiments, the sign or symptom is frequent infections. In certainembodiments, the sign or symptom is tingling or numbness at theextremities. In certain embodiments, the sign or symptom is dry anditchy skin. In certain embodiments, the sign or symptom is weight loss.In certain embodiments, the sign or symptom is slow-healing sores. Incertain embodiments, the sign or symptom is swollen gums. In certainembodiments, the sign or symptom is increased insulin resistance. Incertain embodiments, the sign or symptom is increased glucose levels. Incertain embodiments, the sign or symptom is increased fat mass. Incertain embodiments, the sign or symptom is decreased metabolic rate. Incertain embodiments, the sign or symptom is decreased glucose clearance.In certain embodiments, the sign or symptom is decreased glucosetolerance. In certain embodiments, the sign or symptom is decreasedinsulin sensitivity. In certain embodiments, the sign or symptom isdecreased hepatic insulin sensitivity. In certain embodiments, the signor symptom is increased adipose tissue size and weight. In certainembodiments, the sign or symptom is increased body fat. In certainembodiments, the sign or symptom is increased body weight.

In certain embodiments, provided are methods of treating an individualcomprising administering one or more pharmaceutical compositions asdescribed herein. In certain embodiments, the individual has metabolicrelated disease.

In certain embodiments, administration of a conjugated antisensecompound targeted to a GCCR nucleic acid results in reduction of GCCRexpression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues.

In certain embodiments, pharmaceutical compositions comprising aconjugated antisense compound targeted to GCCR are used for thepreparation of a medicament for treating a patient suffering orsusceptible to metabolic related disease.

In certain embodiments, the methods described herein includeadministering a conjugated compound comprising a modifiedoligonucleotide having a contiguous nucleobases portion as describedherein of a sequence recited in SEQ ID NO: 66-77.

It will be understood that any of the compounds described herein can beused in the aforementioned methods and uses. For example, in certainembodiments a conjugated antisense compound targeted to a GCCR nucleicacid in the aforementioned methods and uses can include, but is notlimited to, a conjugated antisense compound targeted to SEQ ID NO: 7comprising an at least 8 consecutive nucleobase sequence of any one ofSEQ ID NOs: 66-77; a conjugated antisense compound targeted to SEQ IDNO: 7 comprising a nucleobase sequence of any one of SEQ ID NO: 66-77; acompound comprising or consisting of ISIS 420470, ISIS 420476, ISIS426115, ISIS 426130, ISIS 426168, ISIS 426172, ISIS 426183, ISIS 426246,ISIS 426262, ISIS 426267, or ISIS 426325 and a conjugate group; acompound comprising an antisense oligonucleotide disclosed in WO2005/071080, WO 2007/035759, or WO 2007/136988, which are incorporatedby reference in their entireties herein, and a conjugate group; acompound comprising an antisense oligonucleotide having a nucleobasesequence of any of SEQ ID NOs 26-113 disclosed in WO 2007/035759 and aconjugate group described herein; a compound comprising an antisenseoligonucleotide having a nucleobase sequence of any of SEQ ID NOs 26-113disclosed in WO 2007/035759 and a conjugate group described herein; or acompound comprising an antisense oligonucleotide having a nucleobasesequence of any of SEQ ID NOs 413-485 disclosed in WO 2007/136988 and aconjugate group described herein; The nucleobase sequences of all of theaforementioned referenced SEQ ID NOs are incorporated by referenceherein.

4. GCGR

Insulin and glucagon are two pancreatic hormones involved in regulatingglucose homeostasis and metabolism. Glucagon is secreted from theα-cells of the pancreatic islets and regulates glucose homeostasisthrough modulation of hepatic glucose production (Quesada et al., J.Endocrinol. 2008. 199: 5-19). The main function of glucagon is tocounteract the actions of insulin.

Dysregulation of glucose metabolism may be caused either by defectiveinsulin secretion and/or action, or by impaired postprandial glucagonsuppression (Shah et al., Am. J. Physiol. Endocrinol. Metab. 1999. 277:E283-E290). Inhibition of postprandial glucagon secretion in diabeticsubjects has been shown to substantially reduce blood glucose,suggesting that glucagon contributes significantly to the hyperglycemiaseen in subjects with type 2 diabetes mellitus (Shah et al., J. Clin.Endocrinol. Metab. 2000. 85: 4053-4059).

Type 2 diabetes is characterized by impaired insulin secretion and/oraction, and many subjects also exhibit inappropriate levels ofcirculating glucagon in the fasting and postprandial state. An increasein the glucagon/insulin ratio is likely an important determinant of thehyperglycemia seen in type 2 diabetes patients (Baron et al., Diabetes.1987. 36: 274-283). Lack of suppression of postprandial glucagonsecretion in subjects with T2DM also plays an important role in thepathogenesis of postprandial hyperglycemia (Henkel et al., Metabolism.2005. 54: 1168-1173).

Glucagon exerts its action on target tissues via the activation of itsreceptor, GCGR. The glucagon receptor is a 62 kDa protein that is amember of the class B G-protein coupled family of receptors (Brubaker etal., Recept. Channels. 2002. 8: 179-88). GCGR activation leads to signaltransduction by G proteins (G_(s)α and G_(q)), whereby G_(s)α activatesadenylate cyclase, which causes cAMP production, resulting in anincrease in levels of protein kinase A. GCGR signaling in the liverresults in increased hepatic glucose production by induction ofglycogenolysis and gluconeogenesis along with inhibition of glycogenesis(Jiang and Zhang. Am. J. Physiol. Endocrinol. Metab. 2003. 284:E671-E678). GCGR is also expressed in extrahepatic tissues, whichincludes heart, intestinal smooth muscle, kidney, brain, and adiposetissue (Hansen et al., Peptides. 1995. 16: 1163-1166).

Antisense inhibition of GCGR provides a unique advantage overtraditional small molecule inhibitors in that antisense inhibitors donot rely on competitive binding of the compound to the protein andinhibit activity directly by reducing the expression of GCGR. Arepresentative United States patent that teaches GCGR antisenseinhibitors includes U.S. Pat. No. 7,750,142, of which is hereinincorporated by reference in its entirety. Antisense technology isemerging as an effective means for reducing the expression of certaingene products and may therefore prove to be uniquely useful in a numberof therapeutic, diagnostic, and research applications for the modulationof GCGR.

There is a currently a lack of acceptable options for treating metabolicdisorders. It is therefore an object herein to provide compounds andmethods for the treatment of such diseases and disorder. This inventionrelates to the discovery of novel, highly potent inhibitors of GCGR geneexpression. All documents, or portions of documents, cited in thisapplication, including, but not limited to, patents, patentapplications, articles, books, and treatises, are hereby expresslyincorporated-by-reference for the portions of the document discussedherein, as well as in their entirety.

Certain Conjugated Antisense Compounds Targeted to a GCGR Nucleic Acid

In certain embodiments, conjugated antisense compounds are targeted to aGCGR nucleic acid having the sequence GENBANK Accession No. NM_000160.3(incorporated herein as SEQ ID NO: 8) or GENBANK Accession No: NW926918.1 truncated from nucleotides 16865000 to Ser. No. 16/885,000(incorporated herein as SEQ ID NO: 9). In certain such embodiments, aconjugated antisense compound targeted to SEQ ID NOs: 8 and/or 9 is atleast 90%, at least 95%, or 100% complementary to SEQ ID NOs: 8 and/or9.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 8 and/or 9 comprises an at least 8 consecutive nucleobasesequence of any one of SEQ ID NOs: 78-83. In certain embodiments, aconjugated antisense compound targeted to SEQ ID NO: 8 and/or 9comprises a nucleobase sequence of any one of SEQ ID NO: 78-83.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 8 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 78. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 8 comprises a nucleobase sequence of SEQ ID NO:78.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 9 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 79. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 9 comprises a nucleobase sequence of SEQ ID NO:79.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 9 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 80. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 9 comprises a nucleobase sequence of SEQ ID NO:80.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 9 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 81. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 9 comprises a nucleobase sequence of SEQ ID NO:81.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 8 and 9 comprises an at least 8 consecutive nucleobase sequenceof SEQ ID NO: 82. In certain embodiments, a conjugated antisensecompound targeted to SEQ ID NO: 8 and 9 comprises a nucleobase sequenceof SEQ ID NO: 82.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 9 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 83. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 9 comprises a nucleobase sequence of SEQ ID NO:83.

TABLE 5a Antisense Compounds targeted to GCGR SEQ ID NOs: 8 and 9 TargetStart Site to Target Start SEQ ID Site to SEQ SEQ ISIS No NO: 8 ID NO: 9Sequence (5′-3′) Motif ID NO 325568 548 n/a GCACTTTGTGGTGCCAAGGC 2-16-2MOE 78 398471 n/a 8133 TCCACAGGCCACAGGTGGGC 5-10-5 MOE 79 448766 n/a9804 GCAAGGCTCGGTTGGGCTTC 5-10-5 MOE 80 449884 n/a 7270GGTTCCCGAGGTGCCCA 3-10-4 MOE 81 7295 7319 7344 7368 7392 7416 7440459014 227 10718 GGGCAATGCAGTCCTGG 3-10-4 MOE 82 459157 n/a 7267GGGTTCCCGAGGTGCCCAATG 5-10-6 MOE 83 7292 7316 7341 7365 7389 7437

In certain embodiments, a compound comprises or consists of ISIS 325568and a conjugate group. ISIS 325568 is a modified oligonucleotide havingthe formula: Ges mCes Ads mCds Tds Tds Tds Gds Tds Gds Gds Tds Gds mCdsmCds Ads Ads Gds Ges mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 398471and a conjugate group. ISIS 398471 is a modified oligonucleotide havingthe formula: Tes mCes mCes Aes mCes Ads Gds Gds mCds mCds Ads mCds AdsGds Gds Tes Ges Ges Ges mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 448766and a conjugate group. ISIS 448766 is a modified oligonucleotide havingthe formula: Ges mCes Aes Aes Ges Gds mCds Tds mCds Gds Gds Tds Tds GdsGds Ges mCes Tes Tes mCe, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 449884and a conjugate group. ISIS 449884 is a modified oligonucleotide havingthe formula: Ges Ges Tes Tds mCds mCds mCds Gds Ads Gds Gds Tds Gds mCesmCes mCes Ae, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 459014and a conjugate group. ISIS 459014 is a modified oligonucleotide havingthe formula: Ges Ges Ges mCds Ads Ads Tds Gds mCds Ads Gds Tds mCds mCesTes Ges Ge, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 459157and a conjugate group. ISIS 459157 is a modified oligonucleotide havingthe formula: Ges Ges Ges Tes Tes mCds mCds mCds Gds Ads Gds Gds Tds GdsmCds mCes mCes Aes Aes Tes Ge, wherein

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises an antisenseoligonucleotide disclosed in U.S. Pat. No. 7,750,142, U.S. Pat. No.7,399,853, WO 2007/035771, or WO 2007/134014, which are incorporated byreference in their entireties herein, and a conjugate group. In certainembodiments, a compound comprises an antisense oligonucleotide having anucleobase sequence of any of SEQ ID NOs 20-399 disclosed in U.S. Pat.No. 7,750,142 and a conjugate group described herein. In certainembodiments, a compound comprises an antisense oligonucleotide having anucleobase sequence of any of SEQ ID NOs 20-399 disclosed in U.S. Pat.No. 7,399,853 and a conjugate group described herein. In certainembodiments, a compound comprises an antisense oligonucleotide having anucleobase sequence of any of SEQ ID NOs 2 disclosed in WO 2007/035771and a conjugate group described herein. In certain embodiments, acompound comprises an antisense oligonucleotide having a nucleobasesequence of any of SEQ ID NOs: 486-680 disclosed in WO 2007/134014 and aconjugate group described herein. In certain embodiments, a compoundcomprises an antisense oligonucleotide having a nucleobase sequencecomplementary to a preferred target segment of any of SEQ ID NOs 20-399of U.S. Pat. No. 7,750,142, SEQ ID NO: 20-399 of U.S. Pat. No.7,399,853, SEQ ID NO 2 of WO 2007/035771, or SEQ ID NOs. 486-680 of WO2007/134014, and a conjugate group described herein. The nucleobasesequences of all of the aforementioned referenced SEQ ID NOs areincorporated by reference herein.

GCGR Therapeutic Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas metabolic related disease.

As shown in the examples below, conjugated compounds targeted to GCGR,as described herein, have been shown to reduce the severity ofphysiological symptoms of metabolic related diseases, includingmetabolic syndrome, diabetes mellitus, insulin resistance, diabeticdyslipidemia, hypertriglyceridemia, obesity and weight gain. In certainof the experiments, the conjugated compounds reduced blood glucoselevels, e.g., the animals continued to experience symptoms, but thesymptoms were less severe compared to untreated animals. In otherexperiments, however, the conjugated compounds appear to reduce thesymptoms of diabetes; e.g., animals treated for a longer period of timeexperienced less severe symptoms than those administered the compoundsfor a shorter period of time. In other experiments, however, theconjugated compounds appear to inhibit weight gain; e.g., animalstreated for a longer period of time experienced less severe symptomsthan those administered the compounds for a shorter period of time. Inother experiments, however, the conjugated compounds appear to inhibithypertriglyceridemia; e.g., animals treated for a longer period of timeexperienced less severe signs and/or symptoms than those administeredthe compounds for a shorter period of time. The ability of theconjugated compounds exemplified below to restore function thereforedemonstrates that symptoms of the disease may be reversed by treatmentwith a compound as described herein.

Diabetes mellitus is characterized by numerous physical andphysiological signs and/or symptoms. Any symptom known to one of skillin the art to be associated with Type 2 diabetes can be ameliorated orotherwise modulated as set forth above in the methods described above.In certain embodiments, the symptom or sign is a physical symptom orsign such as increased glucose levels, increased weight gain, frequenturination, unusual thirst, extreme hunger, extreme fatigue, blurredvision, frequent infections, tingling or numbness at the extremities,dry and itchy skin, weight loss, slow-healing sores, and swollen gums.In certain embodiments, the symptom or sign is a physiological symptomor sign selected from the group consisting of increased insulinresistance, increased glucose levels, increased fat mass, decreasedmetabolic rate, decreased glucose clearance, decreased glucosetolerance, decreased insulin sensitivity, decreased hepatic insulinsensitivity, increased adipose tissue size and weight, increased bodyfat, and increased body weight.

In certain embodiments, the physical symptom or sign is increasedglucose levels. In certain embodiments, the sign or symptom is weightgain. In certain embodiments, the symptom is frequent urination. Incertain embodiments, the symptom is unusual thirst. In certainembodiments, the symptom is extreme hunger. In certain embodiments, thesymptom is extreme fatigue. In certain embodiments, the symptom isblurred vision. In certain embodiments, the symptom is frequentinfections. In certain embodiments, the symptom is tingling or numbnessat the extremities. In certain embodiments, the symptom is dry and itchyskin. In certain embodiments, the sign or symptom is weight loss. Incertain embodiments, the symptom is slow-healing sores. In certainembodiments, the symptom is swollen gums. In certain embodiments, thesymptom or sign is increased insulin resistance. In certain embodiments,the symptom or sign is increased glucose levels. In certain embodiments,the symptom or sign is increased fat mass. In certain embodiments, thesymptom or sign is decreased metabolic rate. In certain embodiments, thesymptom o rsign is decreased glucose clearance. In certain embodiments,the symptom or sign is decreased glucose tolerance. In certainembodiments, the symptom or sign is decreased insulin sensitivity. Incertain embodiments, the symptom or sign is decreased hepatic insulinsensitivity. In certain embodiments, the symptom or sign is increasedadipose tissue size and weight. In certain embodiments, the symptom orsign is increased body fat. In certain embodiments, the sign or symptomis increased body weight.

In certain embodiments, provided are methods of treating an individualcomprising administering one or more pharmaceutical compositions asdescribed herein. In certain embodiments, the individual has metabolicrelated disease.

In certain embodiments, administration of a conjugated antisensecompound targeted to a GCGR nucleic acid results in reduction of GCGRexpression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues.

In certain embodiments, pharmaceutical compositions comprising aconjugated antisense compound targeted to GCGR are used for thepreparation of a medicament for treating a patient suffering orsusceptible to metabolic related disease.

In certain embodiments, the methods described herein includeadministering a conjugated compound comprising a modifiedoligonucleotide having a contiguous nucleobases portion as describedherein of a sequence recited in SEQ ID NO: 78-83.

It will be understood that any of the compounds described herein can beused in the aforementioned methods and uses. For example, in certainembodiments a conjugated antisense compound targeted to a GCGR nucleicacid in the aforementioned methods and uses can include, but is notlimited to, a conjugated antisense compound targeted to SEQ ID NO: 8and/or 9 comprising an at least 8 consecutive nucleobase sequence of anyone of SEQ ID NOs: 78-83; a conjugated antisense compound targeted toSEQ ID NO: 8 and/or 9 comprising a nucleobase sequence of any one of SEQID NO: 78-83; a compound comprising or consisting of ISIS 325568, ISIS398471, ISIS 448766, ISIS 449884, ISIS 459014, or ISIS 459157 and aconjugate group; a compound comprising an antisense oligonucleotidedisclosed in U.S. Pat. No. 7,750,142, U.S. Pat. No. 7,399,853, WO2007/035771, or WO 2007/134014, which are incorporated by reference intheir entireties herein, and a conjugate group; The nucleobase sequencesof all of the aforementioned referenced SEQ ID NOs are incorporated byreference herein.

4. 4. Coagulation Factor 7

The circulatory system requires mechanisms that prevent blood loss, aswell as those that counteract inappropriate intravascular obstructions.Generally, coagulation comprises a cascade of reactions culminating inthe conversion of soluble fibrinogen to an insoluble fibrin gel. Thesteps of the cascade involve the conversion of an inactive zymogen to anactivated enzyme. The active enzyme then catalyzes the next step in thecascade.

Coagulation Cascade

The coagulation cascade may be initiated through two branches, thetissue factor pathway (also “extrinsic pathway”), which is the primarypathway, and the contact activation pathway (also “intrinsic pathway”).

The tissue factor pathway is initiated by the cell surface receptortissue factor (TF, also referred to as factor III), which is expressedconstitutively by extravascular cells (pericytes, cardiomyocytes, smoothmuscle cells, and keratinocytes) and expressed by vascular monocytes andendothelial cells upon induction by inflammatory cytokines or endotoxin.(Drake et al., Am J Pathol 1989, 134:1087-1097). TF is the high affinitycellular receptor for coagulation factor VIIa, a serine protease. In theabsence of TF, VIIa has very low catalytic activity, and binding to TFis necessary to render VIIa functional through an allosteric mechanism.(Drake et al., Am J Pathol 1989, 134:1087-1097). The TF-VIIa complexactivates factor X to Xa. Xa in turn associates with its co-factorfactor Va into a prothrombinase complex which in turn activatesprothrombin, (also known as factor II or factor 2) to thrombin (alsoknown as factor IIa, or factor 2a). Thrombin activates platelets,converts fibrinogen to fibrin and promotes fibrin cross-linking byactivating factor XIII, thus forming a stable plug at sites where TF isexposed on extravascular cells. In addition, thrombin reinforces thecoagulation cascade response by activating factors V and VIII.

The contact activation pathway is triggered by activation of factor XIIto XIIa. Factor XIIa converts XI to XIa, and XIa converts IX to IXa. IXaassociates with its cofactor Villa to convert X to Xa. The two pathwaysconverge at this point as factor Xa associates factor Va to activateprothrombin (factor II) to thrombin (factor IIa).

Inhibition of Coagulation

At least three mechanisms keep the coagulation cascade in check, namelythe action of activated protein C, antithrombin, and tissue factorpathway inhibitor. Activated protein C is a serine protease thatdegrades cofactors Va and VIIIa. Protein C is activated by thrombin withthrombomodulin, and requires coenzyme Protein S to function.Antithrombin is a serine protease inhibitor (serpin) that inhibitsserine proteases: thrombin, Xa, XIIa, XIa and IXa. Tissue factor pathwayinhibitor inhibits the action of Xa and the TF-VIIa complex. (Schwartz AL et al., Trends Cardiovasc Med. 1997; 7:234-239.)

Disease

Thrombosis is the pathological development of blood clots, and anembolism occurs when a blood clot migrates to another part of the bodyand interferes with organ function. Thromboembolism may cause conditionssuch as deep vein thrombosis, pulmonary embolism, myocardial infarction,and stroke. Significantly, thromboembolism is a major cause of morbidityaffecting over 2 million Americans every year. (Adcock et al. AmericanJournal of Clinical Pathology. 1997; 108:434-49). While most cases ofthrombosis are due to acquired extrinsic problems, for example, surgery,cancer, immobility, some cases are due to a genetic predisposition, forexample, antiphospholipid syndrome and the autosomal dominant condition,Factor V Leiden. (Bertina R M et al. Nature 1994; 369:64-67.)

Treatment

The most commonly used anticoagulants, warfarin, heparin, and lowmolecular weight heparin (LMWH) all possess significant drawbacks.

Warfarin is typically used to treat patients suffering from atrialfibrillation. The drug interacts with vitamin K-dependent coagulationfactors which include factors II, VII, IX and X. Anticoagulant proteinsC and S are also inhibited by warfarin. Drug therapy using warfarin isfurther complicated by the fact that warfarin interacts with othermedications, including drugs used to treat atrial fibrillation, such asamiodarone. Because therapy with warfarin is difficult to predict,patients must be carefully monitored in order to detect any signs ofanomalous bleeding.

Heparin functions by activating antithrombin which inhibits boththrombin and factor X. (Bjork I, Lindahl U. Mol Cell Biochem. 1982 48:161-182.) Treatment with heparin may cause an immunological reactionthat makes platelets aggregate within blood vessels that can lead tothrombosis. This side effect is known as heparin-inducedthrombocytopenia (HIT) and requires patient monitoring. Prolongedtreatment with heparin may also lead to osteoporosis. LMWH can alsoinhibit Factor 2, but to a lesser degree than unfractioned heparin(UFH). LMWH has been implicated in the development of HIT.

Thus, current anticoagulant agents lack predictability and specificityand, therefore, require careful patient monitoring to prevent adverseside effects, such as bleeding complications. There are currently noanticoagulants which target only the intrinsic or extrinsic pathway.

Antisense compounds targeting Factor VII have been previously disclosedin WO 2013/119979 and WO 2009/061851, each herein incorporated byreference in its entirety. Clinical studies are ongoing to assess theeffect of antisense compounds targeting Factor VII in patients. However,there is still a need to provide patients with additional and morepotent treatment options.

Certain Conjugated Antisense Compounds Targeted to a Factor VII NucleicAcid

In certain embodiments, conjugated antisense compounds are targeted to aFactor VII nucleic acid having the sequence GENBANK Accession No.NT_027140.6 truncated from nucleotides 1255000 to 1273000, incorporatedherein as SEQ ID NO: 10; GENBANK Accession No. NM_019616.2, incorporatedherein as SEQ ID NO: 11; DB184141.1, designated herein as SEQ ID NO: 12;and GENBANK Accession No. NW_001104507.1 truncated from nucleotides691000 to 706000, designated herein as SEQ ID NO: 13. In certain suchembodiments, a conjugated antisense compound targeted to SEQ ID NO: 11is at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 11.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 10 comprises an at least 8 consecutive nucleobase sequence of anyone of SEQ ID NOs: 84-92. In certain embodiments, a conjugated antisensecompound targeted to SEQ ID NO: 10 comprises a nucleobase sequence ofany one of SEQ ID NO: 84-92.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 10 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 84. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 10 comprises a nucleobase sequence of SEQ ID NO:84.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 10 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 85. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 10 comprises a nucleobase sequence of SEQ ID NO:85.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 10 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 86. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 10 comprises a nucleobase sequence of SEQ ID NO:86.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 10 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 87. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 10 comprises a nucleobase sequence of SEQ ID NO:87.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 10 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 88. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 10 comprises a nucleobase sequence of SEQ ID NO:88.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 10 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 89. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 10 comprises a nucleobase sequence of SEQ ID NO:89.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 10 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 90. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 10 comprises a nucleobase sequence of SEQ ID NO:90.

TABLE 3a Antisense Compounds targeted to Factor VII SEQ ID NO: 10 TargetStart ISIS No Site Sequence (5′-3′) Motif SEQ ID NO 407935 15191ATGCATGGTGATGCTTCTGA eeeeeddddddddddeeeee 84 473589 15128GCTAAACAACCGCCTT kdkdkdddddddddee 85 490279 1387 CCCTCCTGTGCCTGGATGCTeeeeeddddddddddeeeee 86 529804 15192 CATGGTGATGCTTCTG kddddddddddkekee87 534796 15131 AGAGCTAAACAACCGC ekkddddddddddkke 88 540162 2565; 2633;ACTCCCGGGACACCCA eekddddddddddkke 89 2667 540175 GGACACCCACGCCCCCeekddddddddddkke 90 540182 ACACCCTCGCCTCCGG eekddddddddddkke 91 540191GCCTCCGGAACACCCA eekddddddddddkke 92

In certain embodiments, a compound comprises or consists of ISIS 407935and a conjugate group. ISIS 407935 is a modified oligonucleotide havingthe formula: Aes Tes Ges mCes Aes Tds Gds Gds Tds Gds Ads Tds Gds mCdsTds Tes mCes Tes Ges Ae, wherein

A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises or consists of ISIS 473859and a conjugate group. ISIS 473589 is a modified oligonucleotide havingthe formula: Gks mCds Tks Ads Aks Ads mCds Ads Ads mCds mCds Gds mCdsmCds Tes Te; wherein,

each nucleobase is indicated according to the following:A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises or consists of ISIS 490279and a conjugate group. ISIS 490279 is a modified oligonucleotide havingthe formula: mCes mCes mCes Tes mCes mCds Tds Gds Tds Gds mCds mCds TdsGds Gds Aes Tes Ges mCes Te; wherein,

each nucleobase is indicated according to the following:A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises or consists of ISIS 529804and a conjugate group. ISIS 529804 is a modified oligonucleotide havingthe formula: mCks Ads Tds Gds Gds Tds Gds Ads Tds Gds mCds Tks Tes mCksTes Ge, wherein

A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises or consists of ISIS 534796and a conjugate group. ISIS 534796 is a modified oligonucleotide havingthe formula: Aes Gks Aks Gds mCds Tds Ads Ads Ads mCds Ads Ads mCds mCksGks mCe, wherein

A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises or consists of ISIS 540162and a conjugate group. ISIS 540162 is a modified oligonucleotide havingthe formula: Ges Aes Aes Tes Ges Tds Tds Tds Tds Ads Tds Tds Gds TdsmCds Tes mCes Tes Ges mCe, wherein

A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises or consists of ISIS 540175and a conjugate group. ISIS 540175 is a modified oligonucleotide havingthe formula: Ges Ges Aks mCds Ads mCds mCds mCds Ads mCds Gds mCds mCdsmCks mCks mCe; wherein,

each nucleobase is indicated according to the following:A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises or consists of ISIS 540182and a conjugate group. ISIS 540182 is a modified oligonucleotide havingthe formula: Aes mCes Aks mCds mCds mCds Tds mCds Gds mCds mCds Tds mCdsmCks Gks Ge, wherein

A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises or consists of ISIS 540191and a conjugate group. ISIS 540191 is a modified oligonucleotide havingthe formula: Ges mCes mCks Tds mCds mCds Gds Gds Ads Ads mCds Ads mCdsmCks mCks Ae, wherein

A=adenineT=thymineG=guanine;mC=5-methylcytosine; whereineach sugar moiety is indicated according to the following:k=cEt;d=2′-deoxyribose;e=2′-MOE; whereineach internucleoside linkage is indicated according to the following:s=phosphorothioate.

In certain embodiments, a compound comprises an antisenseoligonucleotide disclosed in WO 2013/119979 and WO 2009/061851, whichare incorporated by reference in their entireties herein. In certainembodiments, a compound comprises an antisense oligonucleotide having anucleobase sequence of any of SEQ ID NOs 21-659 disclosed in WO2013/119979 and a conjugate group described herein. In certainembodiments, a compound comprises an antisense oligonucleotide having anucleobase sequence of any of SEQ ID NOs 4-159 and 168-611 disclosed inWO 2009/061851 and a conjugate group described herein. The nucleobasesequences of all of the aforementioned referenced SEQ ID NOs areincorporated by reference herein.

Factor VII Therapeutic Indications

In certain embodiments, provided herein are compounds and compositionsas described herein for use in therapy.

In certain embodiments, provided herein are conjugated antisensecompounds and compositions as described herein for use in treating,preventing, or slowing progression of a thromboembolic complication.

In certain embodiments, provided herein are conjugated antisensecompounds and compositions as described herein for use in treating,preventing, or slowing progression of a hyperproliferative disorder.

In certain embodiments, provided herein are conjugated antisensecompounds and compositions as described herein for use in treating,preventing, or slowing progression of an inflammatory condition.

In certain embodiments, the invention provides methods for using aconjugated antisense compound targeted to a Factor VII nucleic acid formodulating the expression of Factor VII in a subject. In certainembodiments, the expression of Factor VII is reduced.

In certain embodiments, the invention provides methods for using aconjugated antisense compound targeted to a Factor VII nucleic acid in apharmaceutical composition for treating a subject. In certainembodiments, the subject has a Factor VII related disease, disorder orcondition, or symptom thereof. In certain embodiments, the Factor VIIrelated disease, disorder or condition is a thromboembolic complication,a hyperproliferative disorder or an inflammatory condition.

In certain embodiments, the invention provides methods for using aconjugated antisense compound targeted to a Factor VII nucleic acid inthe preparation of a medicament.

In certain embodiments, the invention provides a conjugated antisensecompound targeted to a Factor VII nucleic acid, or a pharmaceuticallyacceptable salt thereof, for use in therapy.

Certain embodiments provide a conjugated antisense compound targeted toa Factor VII nucleic acid for use in the treatment of a Factor VIIrelated disease, disorder or condition, or symptom thereof. In certainembodiments, the Factor VII related disease, disorder or condition is athromboembolic complication, a hyperproliferative disorder or aninflammatory condition.

In certain embodiments, provided herein are conjugated antisensecompounds and compositions as described herein for use in treating,preventing, or slowing progression of a thromboembolic complication.

In certain embodiments, provided herein are conjugated antisensecompounds and compositions as described herein for use in treating,preventing, or slowing progression of a hyperproliferative disorder.

In certain embodiments, provided herein are conjugated antisensecompounds and compositions as described herein for use in treating,preventing, or slowing progression of an inflammatory condition.

It will be understood that any of the compounds described herein can beused in the aforementioned methods and uses. For example, in certainembodiments a conjugated antisense compound targeted to a Factor VIInucleic acid in the aforementioned methods and uses can include, but isnot limited to, a conjugated antisense compound targeted to SEQ ID NO:10 comprising an at least 8 consecutive nucleobase sequence of any oneof SEQ ID NOs: 84-92; a conjugated antisense compound targeted to SEQ IDNO: 10 comprising a nucleobase sequence of any one of SEQ ID NO: 84-92;a compound comprising or consisting of ISIS 407935, ISIS 473589, ISIS490279, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182,or ISIS 540191 and a conjugate group; a compound comprising an antisenseoligonucleotide disclosed in WO 2013/119979 and WO 2009/061851, whichare incorporated by reference in their entireties herein, and aconjugate group; a compound comprising an antisense oligonucleotidehaving a nucleobase sequence of any of SEQ ID NOs 21-659 disclosed in WO2013/119979 and a conjugate group described herein; an antisenseoligonucleotide having a nucleobase sequence of any of SEQ ID NOs 4-159and 168-611 disclosed in WO 2009/061851 and a conjugate group describedherein. The nucleobase sequences of all of the aforementioned referencedSEQ ID NOs are incorporated by reference herein.

4. Coagulation Factor 11

The circulatory system requires mechanisms that prevent blood loss, aswell as those that counteract inappropriate intravascular obstructions.Generally, coagulation comprises a cascade of reactions culminating inthe conversion of soluble fibrinogen to an insoluble fibrin gel. Thesteps of the cascade involve the conversion of an inactive zymogen to anactivated enzyme. The active enzyme then catalyzes the next step in thecascade.

Coagulation Cascade

The coagulation cascade may be initiated through two branches, thetissue factor pathway (also “extrinsic pathway”), which is the primarypathway, and the contact activation pathway (also “intrinsic pathway”).

The tissue factor pathway is initiated by the cell surface receptortissue factor (TF, also referred to as factor III), which is expressedconstitutively by extravascular cells (pericytes, cardiomyocytes, smoothmuscle cells, and keratinocytes) and expressed by vascular monocytes andendothelial cells upon induction by inflammatory cytokines or endotoxin.(Drake et al., Am J Pathol 1989, 134:1087-1097). TF is the high affinitycellular receptor for coagulation factor VIIa, a serine protease. In theabsence of TF, VIIa has very low catalytic activity, and binding to TFis necessary to render VIIa functional through an allosteric mechanism.(Drake et al., Am J Pathol 1989, 134:1087-1097). The TF-VIIa complexactivates factor X to Xa. Xa in turn associates with its co-factorfactor Va into a prothrombinase complex which in turn activatesprothrombin, (also known as factor II or factor 2) to thrombin (alsoknown as factor IIa, or factor 2a). Thrombin activates platelets,converts fibrinogen to fibrin and promotes fibrin cross-linking byactivating factor XIII, thus forming a stable plug at sites where TF isexposed on extravascular cells. In addition, thrombin reinforces thecoagulation cascade response by activating factors V and VIII.

The contact activation pathway is triggered by activation of factor XIIto XIIa. Factor XIIa converts XI to XIa, and XIa converts IX to IXa. IXaassociates with its cofactor Villa to convert X to Xa. The two pathwaysconverge at this point as factor Xa associates factor Va to activateprothrombin (factor II) to thrombin (factor IIa).

Inhibition of Coagulation.

At least three mechanisms keep the coagulation cascade in check, namelythe action of activated protein C, antithrombin, and tissue factorpathway inhibitor. Activated protein C is a serine protease thatdegrades cofactors Va and VIIIa. Protein C is activated by thrombin withthrombomodulin, and requires coenzyme Protein S to function.Antithrombin is a serine protease inhibitor (serpin) that inhibitsserine proteases: thrombin, Xa, XIIa, XIa and IXa. Tissue factor pathwayinhibitor inhibits the action of Xa and the TF-VIIa complex. (Schwartz AL et al., Trends Cardiovasc Med. 1997; 7:234-239.)

Disease

Thrombosis is the pathological development of blood clots, and anembolism occurs when a blood clot migrates to another part of the bodyand interferes with organ function. Thromboembolism may cause conditionssuch as deep vein thrombosis, pulmonary embolism, myocardial infarction,and stroke. Significantly, thromboembolism is a major cause of morbidityaffecting over 2 million Americans every year. (Adcock et al. AmericanJournal of Clinical Pathology. 1997; 108:434-49). While most cases ofthrombosis are due to acquired extrinsic problems, for example, surgery,cancer, immobility, some cases are due to a genetic predisposition, forexample, antiphospholipid syndrome and the autosomal dominant condition,Factor V Leiden. (Bertina R M et al. Nature 1994; 369:64-67.)

Treatment.

The most commonly used anticoagulants, warfarin, heparin, and lowmolecular weight heparin (LMWH) all possess significant drawbacks.

Warfarin is typically used to treat patients suffering from atrialfibrillation. The drug interacts with vitamin K-dependent coagulationfactors which include factors II, VII, IX and X. Anticoagulant proteinsC and S are also inhibited by warfarin. Drug therapy using warfarin isfurther complicated by the fact that warfarin interacts with othermedications, including drugs used to treat atrial fibrillation, such asamiodarone. Because therapy with warfarin is difficult to predict,patients must be carefully monitored in order to detect any signs ofanomalous bleeding.

Heparin functions by activating antithrombin which inhibits boththrombin and factor X. (Bjork I, Lindahl U. Mol Cell Biochem. 1982 48:161-182.) Treatment with heparin may cause an immunological reactionthat makes platelets aggregate within blood vessels that can lead tothrombosis. This side effect is known as heparin-inducedthrombocytopenia (HIT) and requires patient monitoring. Prolongedtreatment with heparin may also lead to osteoporosis. LMWH can alsoinhibit Factor 2, but to a lesser degree than unfractioned heparin(UFH). LMWH has been implicated in the development of HIT.

Thus, current anticoagulant agents lack predictability and specificityand, therefore, require careful patient monitoring to prevent adverseside effects, such as bleeding complications. There are currently noanticoagulants which target only the intrinsic or extrinsic pathway.

Antisense compounds targeting Factor XI have been previously disclosedin WO 2010/045509 and WO 2010/121074, each herein incorporated byreference in its entirety. Clinical studies are ongoing to assess theeffect of antisense compounds targeting Factor XI in patients. However,there is still a need to provide patients with additional and morepotent treatment options.

Certain Conjugated Antisense Compounds Targeted to a Factor XI NucleicAcid

In certain embodiments, conjugated antisense compounds are targeted to aFactor XI nucleic acid having the sequence GENBANK Accession No.NM_000128.3 (incorporated herein as SEQ ID NO: 14), GENBANK AccessionNo. NT_022792.17, truncated from Ser. No. 19/598,000 to Ser. No.19/624,000, (incorporated herein as SEQ ID NO: 15), GENBANK AccessionNo. NM_028066.1 (incorporated herein as SEQ ID NO: 16), exons 1-15GENBANK Accession No. NW_001118167.1 (incorporated herein as SEQ ID NO:17). In certain such embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 2 is at least 90%, at least 95%, or 100%complementary to SEQ ID NO: 2.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 2 comprises an at least 8 consecutive nucleobase sequence of anyone of SEQ ID NOs: 12-19. In certain embodiments, a conjugated antisensecompound targeted to SEQ ID NO: 2 comprises a nucleobase sequence of anyone of SEQ ID NO: 12-19.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 14 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 93. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 14 comprises a nucleobase sequence of SEQ ID NO:93.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 14 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 94. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 14 comprises a nucleobase sequence of SEQ ID NO:94.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 14 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 95. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 14 comprises a nucleobase sequence of SEQ ID NO:95.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 14 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 96. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 14 comprises a nucleobase sequence of SEQ ID NO:96.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 14 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 97. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 14 comprises a nucleobase sequence of SEQ ID NO:97.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 14 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 98. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 14 comprises a nucleobase sequence of SEQ ID NO:98.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 14 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 99. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 14 comprises a nucleobase sequence of SEQ ID NO:99.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 14 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 100. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 14 comprises a nucleobase sequence of SEQ ID NO:100.

TABLE 7a Antisense Compounds targeted to Factor 11 SEQ ID NO: 14-17Target Start SEQ ID ISIS No Site Sequence (5′-3′) Motif NO 416850TGCACAGTTTCTGGCAGGCC 93 416858 ACGGCATTGGTGCACAGTTT 94 445522GCACAGTTTCTGGCAGGC 95 445531 GGCATTGGTGCACAGTTT 96 449707CACAGTTTCTGGCAGG 97 449708 ACAGTTTCTGGCAG 98 449709 GCACAGTTTCTGGCAGGC99 449710 CACAGTTTCTGGCAGG 100 449711 ACAGTTTCTGGCAG 101

In certain embodiments, a compound comprises an antisenseoligonucleotide disclosed in WO 2010/045509 and WO 2010/121074, whichare incorporated by reference in their entireties herein, and aconjugate group. In certain embodiments, a compound comprises anantisense oligonucleotide having a nucleobase sequence of any of SEQ IDNOs 15-270 disclosed in WO 2010/045509 and a conjugate group describedherein. In certain embodiments, a compound comprises an antisenseoligonucleotide having a nucleobase sequence of any of SEQ ID NOs 15-270disclosed in WO 2010/121074 and a conjugate group described herein. Thenucleobase sequences of all of the aforementioned referenced SEQ ID NOsare incorporated by reference herein.

E. Certain Pharmaceutical Compositions

In certain embodiments, the present disclosure provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligonucleotide which are cleaved by endogenousnucleases within the body, to form the active antisense oligonucleotide.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present disclosureto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present disclosure provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human.

In certain embodiments, the present disclosure provides methods ofadministering a pharmaceutical composition comprising an oligonucleotideof the present disclosure to an animal. Suitable administration routesinclude, but are not limited to, oral, rectal, transmucosal, intestinal,enteral, topical, suppository, through inhalation, intrathecal,intracerebroventricular, intraperitoneal, intranasal, intraocular,intratumoral, and parenteral (e.g., intravenous, intramuscular,intramedullary, and subcutaneous). In certain embodiments,pharmaceutical intrathecals are administered to achieve local ratherthan systemic exposures. For example, pharmaceutical compositions may beinjected directly in the area of desired effect (e.g., into the liver).

NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligonucleotide having the nucleobase sequence “ATCGATCG” encompassesany oligonucleotides having such nucleobase sequence, whether modifiedor unmodified, including, but not limited to, such compounds comprisingRNA bases, such as those having sequence “AUCGAUCG” and those havingsome DNA bases and some RNA bases such as “AUCGATCG” andoligonucleotides having other modified bases, such as “AT^(me)CGAUCG,”wherein ^(me)C indicates a cytosine base comprising a methyl group atthe 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the presentdisclosure and are not limiting. Moreover, where specific embodimentsare provided, the inventors have contemplated generic application ofthose specific embodiments. For example, disclosure of anoligonucleotide having a particular motif provides reasonable supportfor additional oligonucleotides having the same or similar motif. And,for example, where a particular high-affinity modification appears at aparticular position, other high-affinity modifications at the sameposition are considered suitable, unless otherwise indicated.

Example 1: General Method for the Preparation of Phosphoramidites,Compounds 1, 1a and 2

-   -   Bx is a heterocyclic base;

Compounds 1, 1a and 2 were prepared as per the procedures well known inthe art as described in the specification herein (see Seth et al.,Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5),1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); andalso see published PCT International Applications (WO 2011/115818, WO2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO2007/090071), and U.S. Pat. No. 7,569,686).

Example 2: Preparation of Compound 7

Compounds 3(2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-Dgalactopyranose orgalactosamine pentaacetate) is commercially available. Compound 5 wasprepared according to published procedures (Weber et al., J. Med. Chem.,1991, 34, 2692).

Example 3: Preparation of Compound 11

Compounds 8 and 9 are commercially available.

Example 4: Preparation of Compound 18

Compound 11 was prepared as per the procedures illustrated in Example 3.Compound 14 is commercially available. Compound 17 was prepared usingsimilar procedures reported by Rensen et al., J. Med. Chem., 2004, 47,5798-5808.

Example 5: Preparation of Compound 23

Compounds 19 and 21 are commercially available.

Example 6: Preparation of Compound 24

Compounds 18 and 23 were prepared as per the procedures illustrated inExamples 4 and 5.

Example 7: Preparation of Compound 25

Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26

Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9: General Preparation of Conjugated ASOs Comprising GalNAc₃-1at the 3′ Terminus, Compound 29

Wherein the protected GalNAc₃-1 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-1(GalNAc₃-1_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-1_(a) has the formula:

The solid support bound protected GalNAc₃-1, Compound 25, was preparedas per the procedures illustrated in Example 7. Oligomeric Compound 29comprising GalNAc₃-1 at the 3′ terminus was prepared using standardprocedures in automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks,Compounds 1 and 1a were prepared as per the procedures illustrated inExample 1. The phosphoramidites illustrated are meant to berepresentative and not intended to be limiting as other phosphoramiditebuilding blocks can be used to prepare oligomeric compounds having apredetermined sequence and composition. The order and quantity ofphosphoramidites added to the solid support can be adjusted to preparegapped oligomeric compounds as described herein. Such gapped oligomericcompounds can have predetermined composition and base sequence asdictated by any given target.

Example 10: General Preparation Conjugated ASOs Comprising GalNAc₃-1 atthe 5′ Terminus, Compound 34

The Unylinker™ 30 is commercially available. Oligomeric Compound 34comprising a GalNAc₃-1 cluster at the 5′ terminus is prepared usingstandard procedures in automated DNA/RNA synthesis (see Dupouy et al.,Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite buildingblocks, Compounds 1 and 1a were prepared as per the proceduresillustrated in Example 1. The phosphoramidites illustrated are meant tobe representative and not intended to be limiting as otherphosphoramidite building blocks can be used to prepare an oligomericcompound having a predetermined sequence and composition. The order andquantity of phosphoramidites added to the solid support can be adjustedto prepare gapped oligomeric compounds as described herein. Such gappedoligomeric compounds can have predetermined composition and basesequence as dictated by any given target.

Example 11: Preparation of Compound 39

Compounds 4, 13 and 23 were prepared as per the procedures illustratedin Examples 2, 4, and 5. Compound 35 is prepared using similarprocedures published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12,2346-2353.

Example 12: Preparation of Compound 40

Compound 38 is prepared as per the procedures illustrated in Example 11.

Example 13: Preparation of Compound 44

Compounds 23 and 36 are prepared as per the procedures illustrated inExamples 5 and 11. Compound 41 is prepared using similar procedurespublished in WO 2009082607.

Example 14: Preparation of Compound 45

Compound 43 is prepared as per the procedures illustrated in Example 13.

Example 15: Preparation of Compound 47

Compound 46 is commercially available.

Example 16: Preparation of Compound 53

Compounds 48 and 49 are commercially available. Compounds 17 and 47 areprepared as per the procedures illustrated in Examples 4 and 15.

Example 17: Preparation of Compound 54

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 19: General Method for the Preparation of Conjugated ASOsComprising GalNAc₃-1 at the 3′ Position Via Solid Phase Techniques(Preparation of ISIS 647535, 647536 and 651900)

Unless otherwise stated, all reagents and solutions used for thesynthesis of oligomeric compounds are purchased from commercial sources.Standard phosphoramidite building blocks and solid support are used forincorporation nucleoside residues which include for example T, A, G, and^(m)C residues. A 0.1 M solution of phosphoramidite in anhydrousacetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale)or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmolscale) by the phosphoramidite coupling method on an GalNAc₃-1 loadedVIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in thecolumn. For the coupling step, the phosphoramidites were delivered 4fold excess over the loading on the solid support and phosphoramiditecondensation was carried out for 10 min. All other steps followedstandard protocols supplied by the manufacturer. A solution of 6%dichloroacetic acid in toluene was used for removing dimethoxytrityl(DMT) group from 5′-hydroxyl group of the nucleotide.4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activatorduring coupling step. Phosphorothioate linkages were introduced bysulfurization with 0.1 M solution of xanthane hydride in 1:1pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20%tert-butylhydroperoxide in CH₃CN containing 6% water was used as anoxidizing agent to provide phosphodiester internucleoside linkages witha contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphateprotecting groups were deprotected using a 1:1 (v/v) mixture oftriethylamine and acetonitrile with a contact time of 45 minutes. Thesolid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %)and heated at 55° C. for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. Theresidue was purified by high pressure liquid chromatography on a stronganion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm,2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBrin A, 0-40% of B in 60 min, flow 14 mL min-1, λ=260 nm). The residue wasdesalted by HPLC on a reverse phase column to yield the desired ASOs inan isolated yield of 15-30% based on the initial loading on the solidsupport. The ASOs were characterized by ion-pair-HPLC coupled MSanalysis with Agilent 1100 MSD system.

Antisense oligonucleotides not comprising a conjugate were synthesizedusing standard oligonucleotide synthesis procedures well known in theart.

Using these methods, three separate antisense compounds targeting ApoCIII were prepared. As summarized in Table 4, below, each of the threeantisense compounds targeting ApoC III had the same nucleobase sequence;ISIS 304801 is a 5-10-5 MOE gapmer having all phosphorothioate linkages;ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc₃-1conjugated at its 3′end; and ISIS 647536 is the same as ISIS 647535except that certain internucleoside linkages of that compound arephosphodiester linkages. As further summarized in Table 4, two separateantisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages;ISIS 651900 is the same as ISIS 440762, except that it included aGalNAc₃-1 at its 3′-end.

TABLE 4 Modified ASO targeting ApoC III and SRB-1 SEQ CalCd Observed IDASO Sequence (5′ to 3′) Target Mass Mass No. ISIS A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds) T_(es)T_(es)T_(es)A_(es)T_(e) ApoC III7165.4 7164.4 20 304801 ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) - ApoC III 9239.59237.8 21 647535 GalNAc ₃-1 _(a) ISIS A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) ApoC III9142.9 9140.8 21 647536 A _(do′) -GalNAc ₃ -1 _(a) ISIS T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) SRB-1 4647.0 4646.4 22 440762 ISIST_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₃ -1 _(a) SRB-16721.1 6719.4 23 651900Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. “GalNAc₃-1” indicates a conjugate group having thestructure shown previously in Example 9. Note that GalNAc₃-1 comprises acleavable adenosine which links the ASO to remainder of the conjugate,which is designated “GalNAc₃-1_(a).” This nomenclature is used in theabove table to show the full nucleobase sequence, including theadenosine, which is part of the conjugate. Thus, in the above table, thesequences could also be listed as ending with “GalNAc₃-1” with the“A_(do)” omitted. This convention of using the subscript “a” to indicatethe portion of a conjugate group lacking a cleavable nucleoside orcleavable moiety is used throughout these Examples. This portion of aconjugate group lacking the cleavable moiety is referred to herein as a“cluster” or “conjugate cluster” or “GalNAc₃ cluster.” In certaininstances it is convenient to describe a conjugate group by separatelyproviding its cluster and its cleavable moiety.

Example 20: Dose-Dependent Antisense Inhibition of Human ApoC III inhuApoC III Transgenic Mice

ISIS 304801 and ISIS 647535, each targeting human ApoC III and describedabove, were separately tested and evaluated in a dose-dependent studyfor their ability to inhibit human ApoC III in human ApoC III transgenicmice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. ASOs were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. ASOs were dissolved in 0.9% PBS forinjection.

Human ApoC III transgenic mice were injected intraperitoneally once aweek for two weeks with ISIS 304801 or 647535 at 0.08, 0.25. 0.75, 2.25or 6.75 μmol/kg or with PBS as a control. Each treatment group consistedof 4 animals. Forty-eight hours after the administration of the lastdose, blood was drawn from each mouse and the mice were sacrificed andtissues were collected.

ApoC HI mRNA Analysis

ApoC III mRNA levels in the mice's livers were determined usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. ApoC IIImRNA levels were determined relative to total RNA (using Ribogreen),prior to normalization to PBS-treated control. The results below arepresented as the average percent of ApoC III mRNA levels for eachtreatment group, normalized to PBS-treated control and are denoted as “%PBS”. The half maximal effective dosage (ED₅₀) of each ASO is alsopresented in Table 5, below.

As illustrated, both antisense compounds reduced ApoC III RNA relativeto the PBS control. Further, the antisense compound conjugated toGalNAc₃-1 (ISIS 647535) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 5 Effect of ASO treatment on ApoC III mRNA levels in human ApoCIII transgenic mice Dose ED₅₀ SEQ (μmol/ % (μmol/ Internucleoside ID ASOkg) PBS kg) 3′ Conjugate linkage/Length No. PBS 0 100 — — — ISIS 0.08 950.77 None PS/20 20 304801 0.75 42 2.25 32 6.75 19 ISIS 0.08 50 0.074GalNAc ₃ -1 PS/20 21 647535 0.75 15 2.25 17 6.75 8

ApoC III Protein Analysis (Turbidometric Assay)

Plasma ApoC III protein analysis was determined using proceduresreported by Graham et al, Circulation Research, published online beforeprint Mar. 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed withoutdilution using an Olympus Clinical Analyzer and a commercially availableturbidometric ApoC III assay (Kamiya, Cat# KAI-006, Kamiya Biomedical,Seattle, Wash.). The assay protocol was performed as described by thevendor.

As shown in the Table 6 below, both antisense compounds reduced ApoC IIIprotein relative to the PBS control. Further, the antisense compoundconjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent thanthe antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 6 Effect of ASO treatment on ApoC III plasma protein levels inhuman ApoC III transgenic mice Dose ED₅₀ SEQ (μmol/ % (μmol/Internucleoside ID ASO kg) PBS kg) 3′ Conjugate Linkage/Length No. PBS 0100 — — — ISIS 0.08 86 0.73 None PS/20 20 304801 0.75 51 2.25 23 6.75 13ISIS 0.08 72 0.19 GalNAc ₃ -1 PS/20 21 647535 0.75 14 2.25 12 6.75 11

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol.37: 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37,911-917, 1959) (Bligh, E and Dyer, W, Can J Biochem Physiol, 37,911-917, 1959) and measured by using a Beckmann Coulter clinicalanalyzer and commercially available reagents.

The triglyceride levels were measured relative to PBS injected mice andare denoted as “% PBS”. Results are presented in Table 7. Asillustrated, both antisense compounds lowered triglyceride levels.Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535)was substantially more potent than the antisense compound lacking theGalNAc₃-1 conjugate (ISIS 304801).

TABLE 7 Effect of ASO treatment on triglyceride levels in transgenicmice Dose ED₅₀ SEQ (μmol/ % (μmol/ 3′ Internucleoside ID ASO kg) PBS kg)Conjugate Linkage/Length No. PBS 0 100 — — — ISIS 0.08 87 0.63 NonePS/20 20 304801 0.75 46 2.25 21 6.75 12 ISIS 0.08 65 0.13 GalNAc ₃ -1PS/20 21 647535 0.75 9 2.25 8 6.75 9

Plasma samples were analyzed by HPLC to determine the amount of totalcholesterol and of different fractions of cholesterol (HDL and LDL).Results are presented in Tables 8 and 9. As illustrated, both antisensecompounds lowered total cholesterol levels; both lowered LDL; and bothraised HDL. Further, the antisense compound conjugated to GalNAc₃-1(ISIS 647535) was substantially more potent than the antisense compoundlacking the GalNAc₃-1 conjugate (ISIS 304801). An increase in HDL and adecrease in LDL levels is a cardiovascular beneficial effect ofantisense inhibition of ApoC III.

TABLE 8 Effect of ASO treatment on total cholesterol levels intransgenic mice Inter- Total nucleoside Dose Cholesterol 3′ Linkage/ SEQASO (μmol/kg) (mg/dL) Conjugate Length ID No. PBS 0 257 — — ISIS 0.08226 None PS/20 20 304801 0.75 164 2.25 110 6.75 82 ISIS 0.08 230GalNAc₃-1 PS/20 21 647535 0.75 82 2.25 86 6.75 99

TABLE 9 Effect of ASO treatment on HDL and LDL cholesterol levels intransgenic mice Dose HDL SEQ (μmol/ (mg/ LDL 3′ Internucleoside ID ASOkg) dL) (mg/dL) Conjugate Linkage/Length No. PBS 0 17 28 — — ISIS 0.0817 23 None PS/20 32 304801 0.75 27 12 2.25 50 4 6.75 45 2 ISIS 0.08 2121 GalNAc ₃ -1 PS/20 111 647535 0.75 44 2 2.25 50 2 6.75 58 2

Pharmacokinetics Analysis (PK)

The PK of the ASOs was also evaluated. Liver and kidney samples wereminced and extracted using standard protocols. Samples were analyzed onMSD1 utilizing IP-HPLC-MS. The tissue level (μg/g) of full-length ISIS304801 and 647535 was measured and the results are provided in Table 10.As illustrated, liver concentrations of total full-length antisensecompounds were similar for the two antisense compounds. Thus, eventhough the GalNAc₃-1-conjugated antisense compound is more active in theliver (as demonstrated by the RNA and protein data above), it is notpresent at substantially higher concentration in the liver. Indeed, thecalculated EC₅₀ (provided in Table 10) confirms that the observedincrease in potency of the conjugated compound cannot be entirelyattributed to increased accumulation. This result suggests that theconjugate improved potency by a mechanism other than liver accumulationalone, possibly by improving the productive uptake of the antisensecompound into cells.

The results also show that the concentration of GalNAc₃-1 conjugatedantisense compound in the kidney is lower than that of antisensecompound lacking the GalNAc conjugate. This has several beneficialtherapeutic implications. For therapeutic indications where activity inthe kidney is not sought, exposure to kidney risks kidney toxicitywithout corresponding benefit. Moreover, high concentration in kidneytypically results in loss of compound to the urine resulting in fasterclearance. Accordingly, for non-kidney targets, kidney accumulation isundesired. These data suggest that GalNAc₃-1 conjugation reduces kidneyaccumulation.

TABLE 10 PK analysis of ASO treatment in transgenic mice Dose LiverKidney Liver EC₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) (μg/g) (μg/g)(μg/g) Conjugate Linkage/Length ID No. ISIS 0.1 5.2 2.1 53 None PS/20 20304801 0.8 62.8 119.6 2.3 142.3 191.5 6.8 202.3 337.7 ISIS 0.1 3.8 0.73.8 GalNAc ₃ -1 PS/20 21 647535 0.8 72.7 34.3 2.3 106.8 111.4 6.8 237.2179.3

Metabolites of ISIS 647535 were also identified and their masses wereconfirmed by high resolution mass spectrometry analysis. The cleavagesites and structures of the observed metabolites are shown below. Therelative % of full length ASO was calculated using standard proceduresand the results are presented in Table 10a. The major metabolite of ISIS647535 was full-length ASO lacking the entire conjugate (i.e. ISIS304801), which results from cleavage at cleavage site A, shown below.Further, additional metabolites resulting from other cleavage sites werealso observed. These results suggest that introducing other cleabablebonds such as esters, peptides, disulfides, phosphoramidates oracyl-hydrazones between the GalNAc₃-1 sugar and the ASO, which can becleaved by enzymes inside the cell, or which may cleave in the reductiveenvironment of the cytosol, or which are labile to the acidic pH insideendosomes and lyzosomes, can also be useful.

TABLE 10a Observed full length metabolites of ISIS 647535 Metabolite ASOCleavage site Relative % 1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B 10.53 ISIS 647535 minus [3 GalNAc] C 16.1 4 ISIS 647535 minus D 17.6 [3GalNAc + 1 5-hydroxy- pentanoic acid tether] 5 ISIS 647535 minus D 9.9[2 GalNAc + 2 5-hydroxy- pentanoic acid tether] 6 ISIS 647535 minus D9.8 [3 GalNAc + 3 5-hydroxy- pentanoic acid tether]

Example 21: Antisense Inhibition of Human ApoC III in Human ApoC IIITransgenic Mice in Single Administration Study

ISIS 304801, 647535 and 647536 each targeting human ApoC III anddescribed in Table 4, were further evaluated in a single administrationstudy for their ability to inhibit human ApoC III in human ApoC IIItransgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. ASOs were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. ASOs were dissolved in 0.9% PBS forinjection.

Human ApoC III transgenic mice were injected intraperitoneally once atthe dosage shown below with ISIS 304801, 647535 or 647536 (describedabove) or with PBS treated control. The treatment group consisted of 3animals and the control group consisted of 4 animals. Prior to thetreatment as well as after the last dose, blood was drawn from eachmouse and plasma samples were analyzed. The mice were sacrificed 72hours following the last administration.

Samples were collected and analyzed to determine the ApoC III mRNA andprotein levels in the liver; plasma triglycerides; and cholesterol,including HDL and LDL fractions were assessed as described above(Example 20). Data from those analyses are presented in Tables 11-15,below. Liver transaminase levels, alanine aminotransferase (ALT) andaspartate aminotransferase (AST), in serum were measured relative tosaline injected mice using standard protocols. The ALT and AST levelsshowed that the antisense compounds were well tolerated at alladministered doses.

These results show improvement in potency for antisense compoundscomprising a GalNAc₃-1 conjugate at the 3′ terminus (ISIS 647535 and647536) compared to the antisense compound lacking a GalNAc₃-1 conjugate(ISIS 304801). Further, ISIS 647536, which comprises a GalNAc₃-1conjugate and some phosphodiester linkages was as potent as ISIS 647535,which comprises the same conjugate and all internucleoside linkageswithin the ASO are phosphorothioate.

TABLE 11 Effect of ASO treatment on ApoC III mRNA levels in human ApoCIII transgenic mice SEQ Dose % ED₅₀ 3′ Internucleoside ID ASO (mg/kg)PBS (mg/kg) Conjugate linkage/Length No. PBS 0 99 — — — ISIS 1 104 13.2None PS/20 20 304801 3 92 10 71 30 40 ISIS 0.3 98 1.9 GalNAc ₃ -1 PS/2021 647535 1 70 3 33 10 20 ISIS 0.3 103 1.7 GalNAc ₃ -1 PS/PO/20 21647536 1 60 3 31 10 21

TABLE 12 Effect of ASO treatment on ApoC III plasma protein levels inhuman ApoC III transgenic mice ED₅₀ SEQ Dose (mg/ 3′ Internucleoside IDASO (mg/kg) % PBS kg) Conjugate Linkage/Length No. PBS 0 99 — — — ISIS 1104 23.2 None PS/20 20 304801 3 92 10 71 30 40 ISIS 0.3 98 2.1 GalNAc ₃-1 PS/20 21 647535 1 70 3 33 10 20 ISIS 0.3 103 1.8 GalNAc ₃ -1 PS/PO/2021 647536 1 60 3 31 10 21

TABLE 13 Effect of ASO treatment on triglyceride levels in transgenicmice SEQ Dose % ED₅₀ Internucleoside ID ASO (mg/kg) PBS (mg/kg) 3′Conjugate Linkage/Length No. PBS 0 98 — — — ISIS 1 80 29.1 None PS/20 20304801 3 92 10 70 30 47 ISIS 0.3 100 2.2 GalNAc ₃ -1 PS/20 21 647535 170 3 34 10 23 ISIS 0.3 95 1.9 GalNAc ₃ -1 PS/PO/20 21 647536 1 66 3 3110 23

TABLE 14 Effect of ASO treatment on total cholesterol levels intransgenic mice Dose 3′ Internucleoside SEQ ASO (mg/kg) % PBS ConjugateLinkage/Length ID No. PBS 0 96 — — ISIS 1 104 None PS/20 20 304801 3 9610 86 30 72 ISIS 0.3 93 GalNAc₃-1 PS/20 21 647535 1 85 3 61 10 53 ISIS0.3 115 GalNAc₃-1 PS/PO/20 21 647536 1 79 3 51 10 54

TABLE 15 Effect of ASO treatment on HDL and LDL cholesterol levels intransgenic mice LDL SEQ Dose HDL % 3′ Internucleoside ID ASO (mg/kg) %PBS PBS Conjugate Linkage/Length No. PBS 0 131 90 — — ISIS 1 130 72 NonePS/20 20 304801 3 186 79 10 226 63 30 240 46 ISIS 0.3 98 86 GalNAc ₃ -1PS/20 21 647535 1 214 67 3 212 39 10 218 35 ISIS 0.3 143 89 GalNAc ₃ -1PS/PO/20 21 647536 1 187 56 3 213 33 10 221 34

These results confirm that the GalNAc₃-1 conjugate improves potency ofan antisense compound. The results also show equal potency of aGalNAc₃-1 conjugated antisense compounds where the antisenseoligonucleotides have mixed linkages (ISIS 647536 which has sixphosphodiester linkages) and a full phosphorothioate version of the sameantisense compound (ISIS 647535).

Phosphorothioate linkages provide several properties to antisensecompounds. For example, they resist nuclease digestion and they bindproteins resulting in accumulation of compound in the liver, rather thanin the kidney/urine. These are desirable properties, particularly whentreating an indication in the liver. However, phosphorothioate linkageshave also been associated with an inflammatory response. Accordingly,reducing the number of phosphorothioate linkages in a compound isexpected to reduce the risk of inflammation, but also lowerconcentration of the compound in liver, increase concentration in thekidney and urine, decrease stability in the presence of nucleases, andlower overall potency. The present results show that a GalNAc₃-1conjugated antisense compound where certain phosphorothioate linkageshave been replaced with phosphodiester linkages is as potent against atarget in the liver as a counterpart having full phosphorothioatelinkages. Such compounds are expected to be less proinflammatory (SeeExample 24 describing an experiment showing reduction of PS results inreduced inflammatory effect).

Example 22: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting SRB-1In Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 4,were evaluated in a dose-dependent study for their ability to inhibitSRB-1 in Balb/c mice.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900 or with PBS treated control. Each treatment group consisted of 4animals. The mice were sacrificed 48 hours following the finaladministration to determine the SRB-1 mRNA levels in liver usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNAlevels were determined relative to total RNA (using Ribogreen), prior tonormalization to PBS-treated control. The results below are presented asthe average percent of SRB-1 mRNA levels for each treatment group,normalized to PBS-treated control and is denoted as “% PBS”.

As illustrated in Table 16, both antisense compounds lowered SRB-1 mRNAlevels. Further, the antisense compound comprising the GalNAc₃-1conjugate (ISIS 651900) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 440762). These resultsdemonstrate that the potency benefit of GalNAc₃-1 conjugates areobserved using antisense oligonucleotides complementary to a differenttarget and having different chemically modified nucleosides, in thisinstance modified nucleosides comprise constrained ethyl sugar moieties(a bicyclic sugar moiety).

TABLE 16 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c miceLiver SEQ Dose % ED₅₀ Internucleoside ID ASO (mg/kg) PBS (mg/kg) 3′Conjugate linkage/Length No. PBS 0 100 — — ISIS 0.7 85 2.2 None PS/14 22440762 2 55 7 12 20 3 ISIS 0.07 98 0.3 GalNAc ₃ -1 PS/14 23 651900 0.263 0.7 20 2 6 7 5

Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) AssayProtocol

The hPBMC assay was performed using BD Vautainer CPT tube method. Asample of whole blood from volunteered donors with informed consent atUS HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtainedand collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753).The approximate starting total whole blood volume in the CPT tubes foreach donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation bygently inverting tubes 8-10 times. CPT tubes were centrifuged at rt(18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800RCF with brake off (2700 RPM Beckman Allegra 6R). The cells wereretrieved from the buffy coat interface (between Ficoll and polymer gellayers); transferred to a sterile 50 ml conical tube and pooled up to 5CPT tubes/50 ml conical tube/donor. The cells were then washed twicewith PBS (Ca⁺⁺, Mg⁺⁺ free; GIBCO). The tubes were topped up to 50 ml andmixed by inverting several times. The sample was then centrifuged at330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) andaspirated as much supernatant as possible without disturbing pellet. Thecell pellet was dislodged by gently swirling tube and resuspended cellsin RPMI+10% FBS+pen/strep (—1 ml/10 ml starting whole blood volume). A60 μl sample was pipette into a sample vial (Beckman Coulter) with 600μl VersaLyse reagent (Beckman Coulter Cat# A09777) and was gentlyvortexed for 10-15 sec. The sample was allowed to incubate for 10 min.at rt and being mixed again before counting. The cell suspension wascounted on Vicell XR cell viability analyzer (Beckman Coulter) usingPBMC cell type (dilution factor of 1:11 was stored with otherparameters). The live cell/ml and viability were recorded. The cellsuspension was diluted to 1×10⁷ live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵ in 50 μl/well of 96-well tissue cultureplate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controlsdiluted in RPMI+10% FBS+pen/strep. was added according to experimenttemplate (100 μl/well total). Plates were placed on the shaker andallowed to mix for approx. 1 min. After being incubated for 24 hrs at37° C.; 5% CO₂, the plates were centrifuged at 400×g for 10 minutesbefore removing the supernatant for MSD cytokine assay (i.e. human IL-6,IL-10, IL-8 and MCP-1).

Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay forGalNAc₃-1 Conjugated ASOs

The antisense oligonucleotides (ASOs) listed in Table 17 were evaluatedfor proinflammatory effect in hPBMC assay using the protocol describedin Example 23. ISIS 353512 is an internal standard known to be a highresponder for IL-6 release in the assay. The hPBMCs were isolated fromfresh, volunteered donors and were treated with ASOs at 0, 0.0128,0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hrtreatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary readout. The EC₅₀ andE_(max) was calculated using standard procedures. Results are expressedas the average ratio of E_(max)/EC₅₀ from two donors and is denoted as“E_(max)/EC₅₀.” The lower ratio indicates a relative decrease in theproinflammatory response and the higher ratio indicates a relativeincrease in the proinflammatory response.

With regard to the test compounds, the least proinflammatory compoundwas the PS/PO linked ASO (ISIS 616468). The GalNAc₃-1 conjugated ASO,ISIS 647535 was slightly less proinflammatory than its non-conjugatedcounterpart ISIS 304801. These results indicate that incorporation ofsome PO linkages reduces proinflammatory reaction and addition of aGalNAc₃-1 conjugate does not make a compound more proinflammatory andmay reduce proinflammatory response. Accordingly, one would expect thatan antisense compound comprising both mixed PS/PO linkages and aGalNAc₃-1 conjugate would produce lower proinflammatory responsesrelative to full PS linked antisense compound with or without aGalNAc₃-1 conjugate. These results show that GalNAc₃-1 conjugatedantisense compounds, particularly those having reduced PS content areless proinflammatory.

Together, these results suggest that a GalNAc₃-1 conjugated compound,particularly one with reduced PS content, can be administered at ahigher dose than a counterpart full PS antisense compound lacking aGalNAc₃-1 conjugate. Since half-life is not expected to be substantiallydifferent for these compounds, such higher administration would resultin less frequent dosing. Indeed such administration could be even lessfrequent, because the GalNAc₃-1 conjugated compounds are more potent(See Examples 20-22) and re-dosing is necessary once the concentrationof a compound has dropped below a desired level, where such desiredlevel is based on potency.

TABLE 17 Modified ASOs SEQ ID ASO Sequence (5′ to 3′) Target No. ISISG_(es) ^(m)C_(es)T_(es)G_(es)A_(es)T_(ds)T_(ds)A_(ds)G_(ds)A_(ds)G_(ds)TNFα 24 104838 A_(ds)G_(ds)A_(ds)G_(ds)G_(es)T_(es) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) ISIS T_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(ds)A_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) CRP 25 353512G_(ds)A_(ds)G_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es)G_(es)G_(e) ISISA_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)ApoC III 20 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 21 647535 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 20 616468 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. “A_(do′)-GalNAc₃-1_(a)” indicates a conjugate havingthe structure GalNAc₃-1 shown in Example 9 attached to the 3′-end of theantisense oligonucleotide, as indicated.

TABLE 18 Proinflammatory Effect of ASOs targeting ApoC III in hPBMCassay EC₅₀ E_(max) 3′ Internucleoside SEQ ID ASO (μM) (μM) E_(max)/EC₅₀Conjugate Linkage/Length No. ISIS 353512 0.01 265.9 26,590 None PS/20 25(high responder) ISIS 304801 0.07 106.55 1,522 None PS/20 20 ISIS 6475350.12 138 1,150 GalNAc ₃ -1 PS/20 21 ISIS 616468 0.32 71.52 224 NonePS/PO/20 20

Example 25: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting HumanApoC III In Vitro

ISIS 304801 and 647535 described above were tested in vitro. Primaryhepatocyte cells from transgenic mice at a density of 25,000 cells perwell were treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 and 20 μMconcentrations of modified oligonucleotides. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and mRNA levelswere measured by quantitative real-time PCR and the hApoC III mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN.

The IC₅₀ was calculated using the standard methods and the results arepresented in Table 19. As illustrated, comparable potency was observedin cells treated with ISIS 647535 as compared to the control, ISIS304801.

TABLE 19 Modified ASO targeting human ApoC III in primary hepatocytesIC₅₀ 3′ Internucleoside SEQ ASO (μM) Conjugate linkage/Length ID No.ISIS 0.44 None PS/20 20 304801 ISIS 0.31 GalNAc₃-1 PS/20 21 647535

In this experiment, the large potency benefits of GalNAc₃-1 conjugationthat are observed in vivo were not observed in vitro. Subsequent freeuptake experiments in primary hepatocytes in vitro did show increasedpotency of oligonucleotides comprising various GalNAc conjugatesrelative to oligonucleotides that lacking the GalNAc conjugate. (seeExamples 60, 82, and 92)

Example 26: Effect of PO/PS Linkages on ApoC III ASO Activity

Human ApoC III transgenic mice were injected intraperitoneally once at25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or withPBS treated control once per week for two weeks. The treatment groupconsisted of 3 animals and the control group consisted of 4 animals.Prior to the treatment as well as after the last dose, blood was drawnfrom each mouse and plasma samples were analyzed. The mice weresacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III proteinlevels in the liver as described above (Example 20). Data from thoseanalyses are presented in Table 20, below.

These results show reduction in potency for antisense compounds withPO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).

TABLE 20 Effect of ASO treatment on ApoC III protein levels in humanApoC III transgenic mice Dose 3′ Internucleoside SEQ ASO (mg/kg) % PBSConjugate linkage/Length ID No. PBS 0 99 — — ISIS 25 24 None Full PS 20304801 mg/kg/wk for 2 wks ISIS 25 40 None 14 PS/6 PO 20 616468 mg/kg/wkfor 2 wks

Example 27: Compound 56

Compound 56 is commercially available from Glen Research or may beprepared according to published procedures reported by Shchepinov etal., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60

Compound 4 was prepared as per the procedures illustrated in Example 2.Compound 57 is commercially available. Compound 60 was confirmed bystructural analysis.

Compound 57 is meant to be representative and not intended to belimiting as other monoprotected substituted or unsubstituted alkyl diolsincluding but not limited to those presented in the specification hereincan be used to prepare phosphoramidites having a predeterminedcomposition.

Example 29: Preparation of Compound 63

Compounds 61 and 62 are prepared using procedures similar to thosereported by Tober et al., Eur. Org. Chem., 2013, 3, 566-577; and Jianget al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 is prepared using procedures similar to thosereported in scientific and patent literature by Kim et al., Synlett,2003, 12, 1838-1840; and Kim et al., published PCT InternationalApplication, WO 2004063208.

Example 30: Preparation of Compound 63b

Compound 63a is prepared using procedures similar to those reported byHanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31: Preparation of Compound 63d

Compound 63c is prepared using procedures similar to those reported byChen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32: Preparation of Compound 67

Compound 64 was prepared as per the procedures illustrated in Example 2.Compound 65 is prepared using procedures similar to those reported by Oret al., published PCT International Application, WO 2009003009. Theprotecting groups used for Compound 65 are meant to be representativeand not intended to be limiting as other protecting groups including butnot limited to those presented in the specification herein can be used.

Example 33: Preparation of Compound 70

Compound 64 was prepared as per the procedures illustrated in Example 2.Compound 68 is commercially available. The protecting group used forCompound 68 is meant to be representative and not intended to belimiting as other protecting groups including but not limited to thosepresented in the specification herein can be used.

Example 34: Preparation of Compound 75a

Compound 75 is prepared according to published procedures reported byShchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35: Preparation of Compound 79

Compound 76 was prepared according to published procedures reported byShchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36: Preparation of Compound 79a

Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37: General Method for the Preparation of Conjugated OligomericCompound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′Terminus Via Solid Support (Method I)

wherein GalNAc₃-2 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-2(GalNAc₃-2_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-2_(a) has the formula:

The VIMAD-bound oligomeric compound 79b was prepared using standardprocedures for automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56and 60 were prepared as per the procedures illustrated in Examples 27and 28, respectively. The phosphoramidites illustrated are meant to berepresentative and not intended to be limiting as other phosphoramiditebuilding blocks including but not limited those presented in thespecification herein can be used to prepare an oligomeric compoundhaving a phosphodiester linked conjugate group at the 5′ terminus. Theorder and quantity of phosphoramidites added to the solid support can beadjusted to prepare the oligomeric compounds as described herein havingany predetermined sequence and composition.

Example 38: Alternative Method for the Preparation of OligomericCompound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′Terminus (Method II)

The VIMAD-bound oligomeric compound 79b was prepared using standardprocedures for automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). The GalNAc₃-2 clusterphosphoramidite, Compound 79 was prepared as per the proceduresillustrated in Example 35. This alternative method allows a one-stepinstallation of the phosphodiester linked GalNAc₃-2 conjugate to theoligomeric compound at the final step of the synthesis. Thephosphoramidites illustrated are meant to be representative and notintended to be limiting, as other phosphoramidite building blocksincluding but not limited to those presented in the specification hereincan be used to prepare oligomeric compounds having a phosphodiesterconjugate at the 5′ terminus. The order and quantity of phosphoramiditesadded to the solid support can be adjusted to prepare the oligomericcompounds as described herein having any predetermined sequence andcomposition.

Example 39: General Method for the Preparation of Oligomeric Compound83h Comprising a GalNAc₃-3 Conjugate at the 5′ Terminus (GalNAc₃-1Modified for 5′ End Attachment) Via Solid Support

Compound 18 was prepared as per the procedures illustrated in Example 4.Compounds 83a and 83b are commercially available. Oligomeric Compound83e comprising a phosphodiester linked hexylamine was prepared usingstandard oligonucleotide synthesis procedures. Treatment of theprotected oligomeric compound with aqueous ammonia provided the5′-GalNAc₃-3 conjugated oligomeric compound (83h).

Wherein GalNAc₃-3 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-3(GalNAc₃-3_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-3_(a) has the formula:

Example 40: General Method for the Preparation of Oligomeric Compound 89Comprising a Phosphodiester Linked GalNAc₃-4 Conjugate at the 3′Terminus Via Solid Support

Wherein GalNAc₃-4 has the structure:

Wherein CM is a cleavable moiety. In certain embodiments, cleavablemoiety is:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-4(GalNAc₃-4_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-4_(a) has the formula:

The protected Unylinker functionalized solid support Compound 30 iscommercially available. Compound 84 is prepared using procedures similarto those reported in the literature (see Shchepinov et al., NucleicAcids Research, 1997, 25(22), 4447-4454; Shchepinov et al., NucleicAcids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic AcidsResearch, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are preparedas per the procedures illustrated in Examples 28 and 36. Thephosphoramidites illustrated are meant to be representative and notintended to be limiting as other phosphoramidite building blocks can beused to prepare an oligomeric compound having a phosphodiester linkedconjugate at the 3′ terminus with a predetermined sequence andcomposition. The order and quantity of phosphoramidites added to thesolid support can be adjusted to prepare the oligomeric compounds asdescribed herein having any predetermined sequence and composition.

Example 41: General method for the preparation of ASOs comprising aphosphodiester linked GalNAc₃-2 (see Example 37, Bx is adenine)conjugate at the 5′ position via solid phase techniques (preparation ofISIS 661134)

Unless otherwise stated, all reagents and solutions used for thesynthesis of oligomeric compounds are purchased from commercial sources.Standard phosphoramidite building blocks and solid support are used forincorporation nucleoside residues which include for example T, A, G, and^(m)C residues. Phosphoramidite compounds 56 and 60 were used tosynthesize the phosphodiester linked GalNAc₃-2 conjugate at the 5′terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrilewas used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale)or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmolscale) by the phosphoramidite coupling method on VIMAD solid support(110 μmol/g, Guzaev et al., 2003) packed in the column. For the couplingstep, the phosphoramidites were delivered at a 4 fold excess over theinitial loading of the solid support and phosphoramidite coupling wascarried out for 10 min. All other steps followed standard protocolssupplied by the manufacturer. A solution of 6% dichloroacetic acid intoluene was used for removing the dimethoxytrityl (DMT) groups from5′-hydroxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) inanhydrous CH₃CN was used as activator during the coupling step.Phosphorothioate linkages were introduced by sulfurization with 0.1 Msolution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing6% water was used as an oxidizing agent to provide phosphodiesterinternucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphateprotecting groups were deprotected using a 20% diethylamine in toluene(v/v) with a contact time of 45 minutes. The solid-support bound ASOswere suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for6 h. The unbound ASOs were then filtered and the ammonia was boiled off.The residue was purified by high pressure liquid chromatography on astrong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30μm, 2.54×8 cm, λ=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 MNaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, =260 nm). The residuewas desalted by HPLC on a reverse phase column to yield the desired ASOsin an isolated yield of 15-30% based on the initial loading on the solidsupport. The ASOs were characterized by ion-pair-HPLC coupled MSanalysis with Agilent 1100 MSD system.

TABLE 21 ASO comprising a phosphodiester linked GalNAc₃-2 conjugate atthe 5′ position targeting SRB-1 Observed SEQ ID ISIS No. Sequence (5′ to3′) CalCd Mass Mass No. 661134 GalNAc ₃ -2 _(a) - _(o′) A _(do)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) 6482.2 6481.6 26G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. The structure of GalNAc₃-2_(a) is shown in Example37.

Example 42: General Method for the Preparation of ASOs Comprising aGalNAc₃-3 Conjugate at the 5′ Position Via Solid Phase Techniques(Preparation of ISIS 661166)

The synthesis for ISIS 661166 was performed using similar procedures asillustrated in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5′ position comprises aGalNAc₃-3 conjugate. The ASO was characterized by ion-pair-HPLC coupledMS analysis with Agilent 1100 MSD system.

TABLE 21a ASO comprising a GalNAc₃-3 conjugate at the 5′ position via ahexylamino phosphodiester linkage targeting Malat-1 ISIS Calcd ObservedNo. Sequence (5′ to 3′) Conjugate Mass Mass SEQ ID No. 661166 5′-GalNAc₃ -3 _(a-o′) ^(m)C_(es)G_(es)G_(es)T_(es)G_(es) 5′-GalNAc ₃ -3 8992.168990.51 27 ^(m)C_(ds)A_(ds)A_(ds)G_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(ds)A_(ds)G_(ds) G_(es)A_(es)A_(es)T_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “s” indicates phosphorothioateinternucleoside linkages (PS); “o” indicates phosphodiesterinternucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—.Superscript “m” indicates 5-methylcytosines. The structure of“5′-GalNAc₃-3a” is shown in Example 39.

Example 43: Dose-Dependent Study of Phosphodiester Linked GalNAc₃-2 (SeeExamples 37 and 41, Bx is Adenine) at the 5′ Terminus Targeting SRB-1 InVivo

ISIS 661134 (see Example 41) comprising a phosphodiester linkedGalNAc₃-2 conjugate at the 5′ terminus was tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice. Unconjugated ISIS440762 and 651900 (GalNAc₃-1 conjugate at 3′ terminus, see Example 9)were included in the study for comparison and are described previouslyin Table 4.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900, 661134 or with PBS treated control. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the liver SRB-1 mRNA levels usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNAlevels were determined relative to total RNA (using Ribogreen), prior tonormalization to PBS-treated control. The results below are presented asthe average percent of SRB-1 mRNA levels for each treatment group,normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀swere measured using similar methods as described previously and arepresented below.

As illustrated in Table 22, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theantisense oligonucleotides comprising the phosphodiester linkedGalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) or the GalNAc₃-1conjugate linked at the 3′ terminus (ISIS 651900) showed substantialimprovement in potency compared to the unconjugated antisenseoligonucleotide (ISIS 440762). Further, ISIS 661134, which comprises thephosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus wasequipotent compared to ISIS 651900, which comprises the GalNAc₃-1conjugate at the 3′ terminus.

TABLE 22 ASOs containing GalNAc₃-l or GalNAc₃-2 targeting SRB-1 SRB-1mRNA ISIS Dosage levels ED₅₀ SEQ No. (mg/kg) (% PBS) (mg/kg) ConjugateID No. PBS 0 100 — — 440762 0.2 116 2.58 No conjugate 22 0.7 91 2 69 722 20 5 651900 0.07 95 0.26 3′ GalNAc₃-1 23 0.2 77 0.7 28 2 11 7 8661134 0.07 107 0.25 5′ GalNAc₃-2 26 0.2 86 0.7 28 2 10 7 6 Structuresfor 3′ GalNAc₃-1 and 5′ GalNAc₃-2 were described previously in Examples9 and 37.

Pharmacokinetics Analysis (PK)

The PK of the ASOs from the high dose group (7 mg/kg) was examined andevaluated in the same manner as illustrated in Example 20. Liver samplewas minced and extracted using standard protocols. The full lengthmetabolites of 661134 (5′ GalNAc₃-2) and ISIS 651900 (3′ GalNAc₃-1) wereidentified and their masses were confirmed by high resolution massspectrometry analysis. The results showed that the major metabolitedetected for the ASO comprising a phosphodiester linked GalNAc₃-2conjugate at the 5′ terminus (ISIS 661134) was ISIS 440762 (data notshown). No additional metabolites, at a detectable level, were observed.Unlike its counterpart, additional metabolites similar to those reportedpreviously in Table 10a were observed for the ASO having the GalNAc₃-1conjugate at the 3′ terminus (ISIS 651900). These results suggest thathaving the phosphodiester linked GalNAc₃-1 or GalNAc₃-2 conjugate mayimprove the PK profile of ASOs without compromising their potency.

Example 44: Effect of PO/PS Linkages on Antisense Inhibition of ASOsComprising GalNAc₃-1 Conjugate (See Example 9) at the 3′ TerminusTargeting SRB-1

ISIS 655861 and 655862 comprising a GalNAc₃-1 conjugate at the 3′terminus each targeting SRB-1 were tested in a single administrationstudy for their ability to inhibit SRB-1 in mice. The parentunconjugated compound, ISIS 353382 was included in the study forcomparison.

The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten2′-deoxyribonucleosides and each wing region comprises five 2′-MOEmodified nucleosides. The ASOs were prepared using similar methods asillustrated previously in Example 19 and are described Table 23, below.

TABLE 23 Modified ASOs comprising GalNAc₃-1 conjugate at the 3′ terminustargeting SRB-1 SEQ ID ISIS No. Sequence (5′ to 3′) Chemistry No. 353382G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS no conjugate 28 (parent)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS with 29^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate 655862 G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Mixed PS/PO with 29^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) GalNAc ₃-1 conjugate

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “s” indicates phosphorothioateinternucleoside linkages (PS); “o” indicates phosphodiesterinternucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—.Superscript “m” indicates 5-methylcytosines. The structure of“GalNAc₃-1” is shown in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 655862 or with PBS treated control. Each treatment groupconsisted of 4 animals. Prior to the treatment as well as after the lastdose, blood was drawn from each mouse and plasma samples were analyzed.The mice were sacrificed 72 hours following the final administration todetermine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)according to standard protocols. SRB-1 mRNA levels were determinedrelative to total RNA (using Ribogreen), prior to normalization toPBS-treated control. The results below are presented as the averagepercent of SRB-1 mRNA levels for each treatment group, normalized toPBS-treated control and is denoted as “% PBS”. The ED₅₀s were measuredusing similar methods as described previously and are reported below.

As illustrated in Table 24, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner compared to PBStreated control. Indeed, the antisense oligonucleotides comprising theGalNAc₃-1 conjugate at the 3′ terminus (ISIS 655861 and 655862) showedsubstantial improvement in potency comparing to the unconjugatedantisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixedPS/PO linkages showed an improvement in potency relative to full PS(ISIS 655861).

TABLE 24 Effect of PO/PS linkages on antisense inhibition of ASOscomprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISISDosage SRB-1 mRNA ED₅₀ SEQ No. (mg/kg) levels (% PBS) (mg/kg) ChemistryID No. PBS 0 100 — — 353382 3 76.65 10.4 Full PS without 28 (parent) 1052.40 conjugate 30 24.95 655861 0.5 81.22 2.2 Full PS with 29 1.5 63.51GalNAc₃-1 5 24.61 conjugate 15 14.80 655862 0.5 69.57 1.3 Mixed PS/PO 291.5 45.78 with 5 19.70 GalNAc₃-1 15 12.90 conjugate

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Organ weights were alsoevaluated. The results demonstrated that no elevation in transaminaselevels (Table 25) or organ weights (data not shown) were observed inmice treated with ASOs compared to PBS control. Further, the ASO withmixed PS/PO linkages (ISIS 655862) showed similar transaminase levelscompared to full PS (ISIS 655861).

TABLE 25 Effect of PO/PS linkages on transaminase levels of ASOscomprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISISDosage ALT AST No. (mg/kg) (U/L) (U/L) Chemistry SEQ ID No. PBS 0 28.565 — 353382 3 50.25 89 Full PS without 28 (parent) 10 27.5 79.3conjugate 30 27.3 97 655861 0.5 28 55.7 Full PS with 29 1.5 30 78 GalNAc₃ -1 5 29 63.5 15 28.8 67.8 655862 0.5 50 75.5 Mixed PS/PO with 29 1.521.7 58.5 GalNAc ₃ -1 5 29.3 69 15 22 61

Example 45: Preparation of PFP Ester, Compound 110a

Compound 4 (9.5 g, 28.8 mmoles) was treated with compound 103a or 103b(38 mmoles), individually, and TMSOTf (0.5 eq.) and molecular sieves indichloromethane (200 mL), and stirred for 16 hours at room temperature.At that time, the organic layer was filtered thru celite, then washedwith sodium bicarbonate, water and brine. The organic layer was thenseparated and dried over sodium sulfate, filtered and reduced underreduced pressure. The resultant oil was purified by silica gelchromatography (2%->10% methanol/dichloromethane) to give compounds 104aand 104b in >80% yield. LCMS and proton NMR was consistent with thestructure.

Compounds 104a and 104b were treated to the same conditions as forcompounds 100a-d (Example 47), to give compounds 105a and 105b in >90%yield. LCMS and proton NMR was consistent with the structure.

Compounds 105a and 105b were treated, individually, with compound 90under the same conditions as for compounds 901a-d, to give compounds106a (80%) and 106b (20%). LCMS and proton NMR was consistent with thestructure.

Compounds 106a and 106b were treated to the same conditions as forcompounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMSand proton NMR was consistent with the structure.

Compounds 107a and 107b were treated to the same conditions as forcompounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60%yield. LCMS and proton NMR was consistent with the structure.

Compounds 108a (60%) and 108b (40%) were treated to the same conditionsas for compounds 100a-d (Example 47), to give compounds 109a and 109bin >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was treated to the same conditions as for compounds 101a-d(Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMRwas consistent with the structure. Alternatively, Compound 110b can beprepared in a similar manner starting with Compound 109b.

Example 46: General Procedure for Conjugation with PFP Esters(Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc₃-10)

A 5′-hexylamino modified oligonucleotide was synthesized and purifiedusing standard solid-phase oligonucleotide procedures. The 5′-hexylaminomodified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH8.5 (200 μL) and 3 equivalents of a selected PFP esterified GalNAc₃cluster dissolved in DMSO (50 μL) was added. If the PFP esterprecipitated upon addition to the ASO solution DMSO was added until allPFP ester was in solution. The reaction was complete after about 16 h ofmixing at room temperature. The resulting solution was diluted withwater to 12 mL and then spun down at 3000 rpm in a spin filter with amass cut off of 3000 Da. This process was repeated twice to remove smallmolecule impurities. The solution was then lyophilized to dryness andredissolved in concentrated aqueous ammonia and mixed at roomtemperature for 2.5 h followed by concentration in vacuo to remove mostof the ammonia. The conjugated oligonucleotide was purified and desaltedby RP-HPLC and lyophilized to provide the GalNAc₃ conjugatedoligonucleotide.

Oligonucleotide 111 is conjugated with GalNAc₃-10. The GalNAc₃ clusterportion of the conjugate group GalNAc₃-10 (GalNAc₃-10_(a)) can becombined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is —P(═O)(OH)—A_(d)-P(═O)(OH)— as shown in the oligonucleotide (ISIS 666881)synthesized with GalNAc₃-10 below. The structure of GalNAc₃-10(GalNAc₃-10_(a)-CM-) is shown below:

Following this general procedure ISIS 666881 was prepared. 5′-hexylaminomodified oligonucleotide, ISIS 660254, was synthesized and purifiedusing standard solid-phase oligonucleotide procedures. ISIS 660254 (40mg, 5.2 μmop was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL)and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 μL)was added. The PFP ester precipitated upon addition to the ASO solutionrequiring additional DMSO (600 μL) to fully dissolve the PFP ester. Thereaction was complete after 16 h of mixing at room temperature. Thesolution was diluted with water to 12 mL total volume and spun down at3000 rpm in a spin filter with a mass cut off of 3000 Da. This processwas repeated twice to remove small molecule impurities. The solution waslyophilized to dryness and redissolved in concentrated aqueous ammoniawith mixing at room temperature for 2.5 h followed by concentration invacuo to remove most of the ammonia. The conjugated oligonucleotide waspurified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in90% yield by weight (42 mg, 4.7 μmol).

GalNAc₃-10 conjugated oligonucleotide SEQ ASO Sequence (5′ to 3′)5′ group ID No. ISIS 660254 NH₂(CH₂)₆-_(o)A_(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) Hexylamine 30^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) GalNAc ₃-10 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc₃-8

The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) andN,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyltrifluoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon,and the reaction was allowed to stir at room temperature for 30 minutes.Boc-diamine 91a or 91b (68.87 mmol) was added, along withN,N-Diisopropylethylamine (12.35 mL, 72 mmoles), and the reaction wasallowed to stir at room temperature for 16 hours. At that time, the DMFwas reduced by >75% under reduced pressure, and then the mixture wasdissolved in dichloromethane. The organic layer was washed with sodiumbicarbonate, water and brine. The organic layer was then separated anddried over sodium sulfate, filtered and reduced to an oil under reducedpressure. The resultant oil was purified by silica gel chromatography(2%->10% methanol/dichloromethane) to give compounds 92a and 92b in anapproximate 80% yield. LCMS and proton NMR were consistent with thestructure.

Compound 92a or 92b (6.7 mmoles) was treated with 20 mL ofdichloromethane and 20 mL of trifluoroacetic acid at room temperaturefor 16 hours. The resultant solution was evaporated and then dissolvedin methanol and treated with DOWEX-OH resin for 30 minutes. Theresultant solution was filtered and reduced to an oil under reducedpressure to give 85-90% yield of compounds 93a and 93b.

Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7 g, 9.6mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15minutes. To this was added either compounds 93a or 93b (3 mmoles), andallowed to stir at room temperature for 16 hours. At that time, the DMFwas reduced by >75% under reduced pressure, and then the mixture wasdissolved in dichloromethane. The organic layer was washed with sodiumbicarbonate, water and brine. The organic layer was then separated anddried over sodium sulfate, filtered and reduced to an oil under reducedpressure. The resultant oil was purified by silica gel chromatography(5%->20% methanol/dichloromethane) to give compounds 96a-d in 20-40%yield. LCMS and proton NMR was consistent with the structure.

Compounds 96a-d (0.75 mmoles), individually, were hydrogenated overRaney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalystwas removed by filtration thru celite, and the ethanol removed underreduced pressure to give compounds 97a-d in 80-90% yield. LCMS andproton NMR were consistent with the structure.

Compound 23 (0.32 g, 0.53 mmoles) was treated with HBTU (0.2 g, 0.53mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles),individually, and allowed to stir at room temperature for 16 hours. Atthat time, the DMF was reduced by >75% under reduced pressure, and thenthe mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (2%->20% methanol/dichloromethane) to give compounds98a-d in 30-40% yield. LCMS and proton NMR was consistent with thestructure.

Compound 99 (0.17 g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles),individually, and allowed to stir at room temperature for 16 hours. Atthat time, the DMF was reduced by >75% under reduced pressure, and thenthe mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (5%->20% methanol/dichloromethane) to give compounds100a-d in 40-60% yield. LCMS and proton NMR was consistent with thestructure.

Compounds 100a-d (0.16 mmoles), individually, were hydrogenated over 10%Pd(OH)₂/C for 3 hours in methanol/ethyl acetate (1:1, 50 mL). At thattime, the catalyst was removed by filtration thru celite, and theorganics removed under reduced pressure to give compounds 101a-d in80-90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15mL) and pyridine (0.016 mL, 0.2 mmoles). Pentafluorophenyltrifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon,and the reaction was allowed to stir at room temperature for 30 minutes.At that time, the DMF was reduced by >75% under reduced pressure, andthen the mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (2%->5% methanol/dichloromethane) to give compounds102a-d in an approximate 80% yield. LCMS and proton NMR were consistentwith the structure.

Oligomeric Compound 102, comprising a GalNAc₃-8 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-8 (GalNAc₃-8_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In a preferred embodiment, the cleavable moiety is—P(═O)(OH)— A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-8 (GalNAc₃-8_(a)-CM-) is shown below:

Example 48: Preparation of Oligonucleotide 119 Comprising GalNAc₃-7

Compound 112 was synthesized following the procedure described in theliterature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate(22 mL/22 mL). Palladium hydroxide on carbon (0.5 g) was added. Thereaction mixture was stirred at room temperature under hydrogen for 12h. The reaction mixture was filtered through a pad of celite and washedthe pad with 1:1 methanol/ethyl acetate. The filtrate and the washingswere combined and concentrated to dryness to yield Compound 105a(quantitative). The structure was confirmed by LCMS.

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reactionmixture was stirred at room temperature for 5 min. To this a solution ofCompound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. Thereaction was stirred at room temperature for 6 h. Solvent was removedunder reduced pressure to get an oil. The residue was dissolved inCH₂Cl₂ (100 mL) and washed with aqueous saturated NaHCO₃ solution (100mL) and brine (100 mL). The organic phase was separated, dried (Na₂SO₄),filtered and evaporated. The residue was purified by silica gel columnchromatography and eluted with 10 to 20% MeOH in dichloromethane toyield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMSand ¹H NMR analysis.

Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethylacetate (4 mL/4 mL). Palladium on carbon (wet, 0.14 g) was added. Thereaction mixture was flushed with hydrogen and stirred at roomtemperature under hydrogen for 12 h. The reaction mixture was filteredthrough a pad of celite. The celite pad was washed with methanol/ethylacetate (1:1). The filtrate and the washings were combined together andevaporated under reduced pressure to yield Compound 115 (quantitative).The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA(0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and thereaction mixture was stirred at room temperature for 5 min. To this asolution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was addedand the reaction was stirred at room temperature for 6 h. The solventwas removed under reduced pressure and the residue was dissolved inCH₂Cl₂. The organic layer was washed aqueous saturated NaHCO₃ solutionand brine and dried over anhydrous Na₂SO₄ and filtered. The organiclayer was concentrated to dryness and the residue obtained was purifiedby silica gel column chromatography and eluted with 3 to 15% MeOH indichloromethane to yield Compound 116 (0.84 g, 61%). The structure wasconfirmed by LC MS and ¹H NMR analysis.

Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethylacetate (5 mL/5 mL). Palladium on carbon (wet, 0.074 g) was added. Thereaction mixture was flushed with hydrogen and stirred at roomtemperature under hydrogen for 12 h. The reaction mixture was filteredthrough a pad of celite. The celite pad was washed with methanol/ethylacetate (1:1). The filtrate and the washings were combined together andevaporated under reduced pressure to yield compound 117 (0.73 g, 98%).The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL).To this solution N,N-Diisopropylethylamine (70 μL, 0.4 mmol) andpentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. Thereaction mixture was stirred at room temperature for 12 h and pouredinto a aqueous saturated NaHCO₃ solution. The mixture was extracted withdichloromethane, washed with brine and dried over anhydrous Na₂SO₄. Thedichloromethane solution was concentrated to dryness and purified withsilica gel column chromatography and eluted with 5 to 10% MeOH indichloromethane to yield compound 118 (0.51 g, 79%). The structure wasconfirmed by LCMS and ¹H and ¹H and ¹⁹F NMR.

Oligomeric Compound 119, comprising a GalNAc₃-7 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-7 (GalNAc₃-7_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)— A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-7 (GalNAc₃-7_(a)-CM-) is shown below:

Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc₃-5

Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) weredissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol)was added and stirred for 5 min. The reaction mixture was cooled in anice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF(20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) wasadded and the reaction mixture was stirred for 18 h under an argonatmosphere. The reaction was monitored by TLC (ethyl acetate:hexane;1:1; Rf=0.47). The solvent was removed under reduced pressure. Theresidue was taken up in EtOAc (300 mL) and washed with 1M NaHSO₄ (3×150mL), aqueous saturated NaHCO₃ solution (3×150 mL) and brine (2×100 mL).Organic layer was dried with Na₂SO₄. Drying agent was removed byfiltration and organic layer was concentrated by rotary evaporation.Crude mixture was purified by silica gel column chromatography andeluted by using 35-50% EtOAc in hexane to yield a compound 122 (15.50 g,78.13%). The structure was confirmed by LCMS and ¹H NMR analysis. Massm/z 589.3 [M+H]⁺.

A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) wasadded to a cooled solution of Compound 122 (7.75 g, 13.16 mmol)dissolved in methanol (15 mL). The reaction mixture was stirred at roomtemperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). Thereaction mixture was concentrated to half the volume under reducedpressure. The remaining solution was cooled an ice bath and neutralizedby adding concentrated HCl. The reaction mixture was diluted, extractedwith EtOAc (120 mL) and washed with brine (100 mL). An emulsion formedand cleared upon standing overnight. The organic layer was separateddried (Na₂SO₄), filtered and evaporated to yield Compound 123 (8.42 g).Residual salt is the likely cause of excess mass. LCMS is consistentwith structure. Product was used without any further purification. M.W.cal:574.36; M.W. fd:575.3 [M+H]⁺.

Compound 126 was synthesized following the procedure described in theliterature (J. Am. Chem. Soc. 2011, 133, 958-963).

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) andcompound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) andthe resulting reaction mixture was cooled in an ice bath. To thisN,N-Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were addedunder an argon atmosphere. The ice bath was removed and the solution wasallowed to warm to room temperature. The reaction was completed after 1h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture wasconcentrated under reduced pressure. The residue was dissolved in EtOAc(200 mL) and washed with 1 M NaHSO₄ (3×100 mL), aqueous saturated NaHCO₃(3×100 mL) and brine (2×100 mL). The organic phase separated dried(Na₂SO₄), filtered and concentrated. The residue was purified by silicagel column chromatography with a gradient of 50% hexanes/EtOAC to 100%EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and ¹H NMRwere consistent with structure. Mass m/z 778.4 [M+H]+.

Trifluoroacetic acid (12 mL) was added to a solution of compound 127(1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at roomtemperature for 1 h. The reaction mixture was co-evaporated with toluene(30 mL) under reduced pressure to dryness. The residue obtained wasco-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) toyield Compound 128 (1.67 g) as trifluoro acetate salt and used for nextstep without further purification. LCMS and ¹H NMR were consistent withstructure. Mass m/z 478.2 [M+H]+.

Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt(0.035 g, 0.26 mmol) were combined together and dried for 4 h over P₂O₅under reduced pressure in a round bottom flask and then dissolved inanhydrous DMF (1 mL) and stirred for 5 min. To this a solution ofcompound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) andN,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture wasstirred at room temperature under an argon atmosphere. The reaction wascomplete after 30 min as determined by LCMS and TLC (7% MeOH/DCM). Thereaction mixture was concentrated under reduced pressure. The residuewas dissolved in DCM (30 mL) and washed with 1 M NaHSO₄ (3×20 mL),aqueous saturated NaHCO₃ (3×20 mL) and brine (3×20 mL). The organicphase was separated, dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by silica gel column chromatography using 5-15%MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and ¹HNMR are consistent with structure. Mass m/z 883.4 [M+2H]⁺.

Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in 20mL scintillation vial. To this was added a small amount of 10% Pd/C(0.015 mg) and the reaction vessel was flushed with H₂ gas. The reactionmixture was stirred at room temperature under H₂ atmosphere for 18 h.The reaction mixture was filtered through a pad of Celite and the Celitepad was washed with methanol. The filtrate washings were pooled togetherand concentrated under reduced pressure to yield Compound 130 (0.08 g).LCMS and ¹H NMR were consistent with structure. The product was usedwithout further purification. Mass m/z 838.3 [M+2H]⁺.

To a 10 mL pointed round bottom flask were added compound 130 (75.8 mg,0.046 mmol), 0.37 M pyridine/DMF (200 μL) and a stir bar. To thissolution was added 0.7 M pentafluorophenyl trifluoroacetate/DMF (100 μL)drop wise with stirring. The reaction was completed after 1 h asdetermined by LC MS. The solvent was removed under reduced pressure andthe residue was dissolved in CHCl₃ (˜10 mL). The organic layer waspartitioned against NaHSO₄ (1 M, 10 mL), aqueous saturated NaHCO₃ (10mL) and brine (10 mL) three times each. The organic phase separated anddried over Na₂SO₄, filtered and concentrated to yield Compound 131 (77.7mg). LCMS is consistent with structure. Used without furtherpurification. Mass m/z 921.3 [M+2H]⁺.

Oligomeric Compound 132, comprising a GalNAc₃-5 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-5 (GalNAc₃-5_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)— A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-5 (GalNAc₃-5_(a)-CM-) is shown below:

Example 50: Preparation of Oligonucleotide 144 Comprising GalNAc4-11

Synthesis of Compound 134. To a Merrifield flask was added aminomethylVIMAD resin (2.5 g, 450 μmol/g) that was washed with acetonitrile,dimethylformamide, dichloromethane and acetonitrile. The resin wasswelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol,0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solutionwas allowed to stir for 5 min and was then added to the Merrifield flaskwith shaking. The suspension was allowed to shake for 3 h. The reactionmixture was drained and the resin was washed with acetonitrile, DMF andDCM. New resin loading was quantitated by measuring the absorbance ofthe DMT cation at 500 nm (extinction coefficient=76000) in DCM anddetermined to be 238 μmol/g. The resin was capped by suspending in anacetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was synthesized using iterativeFmoc-based solid phase peptide synthesis methods. A small amount ofsolid support was withdrawn and suspended in aqueous ammonia (28-30 wt%) for 6 h. The cleaved compound was analyzed by LC-MS and the observedmass was consistent with structure. Mass m/z 1063.8 [M+2H]⁺.

The solid support bound compound 142 was synthesized using solid phasepeptide synthesis methods.

The solid support bound compound 143 was synthesized using standardsolid phase synthesis on a DNA synthesizer.

The solid support bound compound 143 was suspended in aqueous ammonia(28-30 wt %) and heated at 55° C. for 16 h. The solution was cooled andthe solid support was filtered. The filtrate was concentrated and theresidue dissolved in water and purified by HPLC on a strong anionexchange column. The fractions containing full length compound 144 werepooled together and desalted. The resulting GalNAc4-11 conjugatedoligomeric compound was analyzed by LC-MS and the observed mass wasconsistent with structure.

The GalNAc4 cluster portion of the conjugate group GalNAc4-11(GalNAc4-11_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)— A_(d)-P(═O)(OH)—.

The structure of GalNAc4-11 (GalNAc4-11_(a)-CM) is shown below:

Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc₃-6

Compound 146 was synthesized as described in the literature (AnalyticalBiochemistry 1995, 229, 54-60).

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol)were dissolved in CH₂Cl₂ (200 ml). Activated molecular sieves (4 Å. 2 g,powdered) were added, and the reaction was allowed to stir for 30minutes under nitrogen atmosphere. TMS-OTf was added (4.1 ml, 22.77mmol) and the reaction was allowed to stir at room temp overnight. Uponcompletion, the reaction was quenched by pouring into solution ofsaturated aqueous NaHCO₃ (500 ml) and crushed ice (˜150 g). The organiclayer was separated, washed with brine, dried over MgSO₄, filtered, andwas concentrated to an orange oil under reduced pressure. The crudematerial was purified by silica gel column chromatography and elutedwith 2-10% MeOH in CH₂Cl₂ to yield Compound 112 (16.53 g, 63%). LCMS and¹H NMR were consistent with the expected compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40ml). The reaction mixture was purged by bubbling a stream of argonthrough the solution for 15 minutes. Pearlman's catalyst (palladiumhydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbledthrough the solution for 30 minutes. Upon completion (TLC 10% MeOH inCH₂Cl₂, and LCMS), the catalyst was removed by filtration through a padof celite. The filtrate was concentrated by rotary evaporation, and wasdried briefly under high vacuum to yield Compound 105a (3.28 g). LCMSand 1H NMR were consistent with desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL).N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed byHBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ˜15minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4mmol) in dry DMF was added and stirred for 2 h under nitrogenatmosphere. The reaction was diluted with EtOAc and washed withsaturated aqueous NaHCO₃ and brine. The organics phase was separated,dried (MgSO₄), filtered, and concentrated to an orange syrup. The crudematerial was purified by column chromatography 2-5% MeOH in CH₂Cl₂ toyield Compound 148 (3.44 g, 73%). LCMS and ¹H NMR were consistent withthe expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml).The reaction mixture was purged by bubbling a stream of argon throughthe solution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (350 mg). Hydrogen gas was bubbled through thesolution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, andLCMS), the catalyst was removed by filtration through a pad of celite.The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 149 (2.6 g). LCMS wasconsistent with desired product. The residue was dissolved in dry DMF(10 ml) was used immediately in the next step.

Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). Tothis DIEA (450 μL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) wereadded. The reaction mixture was allowed to stir for 15 minutes at roomtemperature under nitrogen. A solution of compound 149 (2.6 g) inanhydrous DMF (10 mL) was added. The pH of the reaction was adjusted topH=9-10 by addition of DIEA (if necessary). The reaction was allowed tostir at room temperature under nitrogen for 2 h. Upon completion thereaction was diluted with EtOAc (100 mL), and washed with aqueoussaturated aqueous NaHCO₃, followed by brine. The organic phase wasseparated, dried over MgSO₄, filtered, and concentrated. The residue waspurified by silica gel column chromatography and eluted with 2-10% MeOHin CH₂Cl₂ to yield Compound 150 (0.62 g, 20%). LCMS and ¹H NMR wereconsistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L). Thereaction mixture was purged by bubbling a stream of argon through thesolution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (60 mg). Hydrogen gas was bubbled through the solutionfor 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), thecatalyst was removed by filtration (syringe tip Teflon filter, 0.45 μm).The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 151 (0.57 g). The LCMS wasconsistent with the desired product. The product was dissolved in 4 mLdry DMF and was used immediately in the next step.

Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL)and N,N-Diisopropylethylamine (75 μL, 1 mmol) and PFP-TFA (90 μL, 0.76mmol) were added. The reaction mixture turned magenta upon contact, andgradually turned orange over the next 30 minutes. Progress of reactionwas monitored by TLC and LCMS. Upon completion (formation of the PFPester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added.The pH of the reaction was adjusted to pH=9-10 by addition ofN,N-Diisopropylethylamine (if necessary). The reaction mixture wasstirred under nitrogen for ˜30 min. Upon completion, the majority of thesolvent was removed under reduced pressure. The residue was diluted withCH₂Cl₂ and washed with aqueous saturated NaHCO₃, followed by brine. Theorganic phase separated, dried over MgSO₄, filtered, and concentrated toan orange syrup. The residue was purified by silica gel columnchromatography (2-10% MeOH in CH₂Cl₂) to yield Compound 152 (0.35 g,55%). LCMS and ¹H NMR were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10mL). The reaction mixture was purged by bubbling a stream of argon thruthe solution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (35 mg). Hydrogen gas was bubbled thru the solutionfor 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), thecatalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm).The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 153 (0.33 g, quantitative).The LCMS was consistent with desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL)with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 μL,0.37 mmol) and PFP-TFA (35 μL, 0.28 mmol) were added. The reactionmixture was stirred under nitrogen for ˜30 min. The reaction mixtureturned magenta upon contact, and gradually turned orange. The pH of thereaction mixture was maintained at pH=9-10 by adding moreN,-Diisopropylethylamine. The progress of the reaction was monitored byTLC and LCMS. Upon completion, the majority of the solvent was removedunder reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL), andwashed with saturated aqueous NaHCO₃, followed by brine. The organiclayer was dried over MgSO₄, filtered, and concentrated to an orangesyrup. The residue was purified by column chromatography and eluted with2-10% MeOH in CH₂Cl₂ to yield Compound 154 (0.29 g, 79%). LCMS and ¹HNMR were consistent with the desired product.

Oligomeric Compound 155, comprising a GalNAc₃-6 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-6 (GalNAc₃-6_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)— A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-6 (GalNAc₃-6_(a)-CM-) is shown below:

Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc₃-9

Compound 156 was synthesized following the procedure described in theliterature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL).Palladium on carbon (6.15 g, 10 wt %, loading (dry basis), matrix carbonpowder, wet) was added. The reaction mixture was stirred at roomtemperature under hydrogen for 18 h. The reaction mixture was filteredthrough a pad of celite and the celite pad was washed thoroughly withmethanol. The combined filtrate was washed and concentrated to dryness.The residue was purified by silica gel column chromatography and elutedwith 5-10% methanol in dichloromethane to yield Compound 157 (14.26 g,89%). Mass m/z 544.1 [M−H]⁻.

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL).HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81mmol) were added and the reaction mixture was stirred at roomtemperature for 5 minutes. To this a solution of compound 47 (2.96 g,7.04 mmol) was added. The reaction was stirred at room temperature for 8h. The reaction mixture was poured into a saturated NaHCO₃ aqueoussolution. The mixture was extracted with ethyl acetate and the organiclayer was washed with brine and dried (Na₂SO₄), filtered and evaporated.The residue obtained was purified by silica gel column chromatographyand eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25g, 73.3%). The structure was confirmed by MS and ¹H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P₂O₅ under reducedpressure. The dried compound was dissolved in anhydrous DMF (50 mL). Tothis 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 mL,3.81 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite(3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred tunder an argon atmosphere for 4 h. The reaction mixture was diluted withethyl acetate (200 mL). The reaction mixture was washed with saturatedNaHCO₃ and brine. The organic phase was separated, dried (Na₂SO₄),filtered and evaporated. The residue was purified by silica gel columnchromatography and eluted with 50-90% ethyl acetate in hexane to yieldCompound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and³¹P NMR analysis.

Oligomeric Compound 160, comprising a GalNAc₃-9 conjugate group, wasprepared using standard oligonucleotide synthesis procedures. Threeunits of compound 159 were coupled to the solid support, followed bynucleotide phosphoramidites. Treatment of the protected oligomericcompound with aqueous ammonia yielded compound 160. The GalNAc₃ clusterportion of the conjugate group GalNAc₃-9 (GalNAc₃-9_(a)) can be combinedwith any cleavable moiety to provide a variety of conjugate groups. Incertain embodiments, the cleavable moiety is —P(═O)(OH)—A_(d)-P(═O)(OH)—. The structure of GalNAc₃-9 (GalNAc₃-9_(a)-CM) is shownbelow:

Example 53: Alternate Procedure for Preparation of Compound 18(GalNAc₃-1a and GalNAc₃-3a)

Lactone 161 was reacted with diamino propane (3-5 eq) or Mono-Bocprotected diamino propane (1 eq) to provide alcohol 162a or 162b. Whenunprotected propanediamine was used for the above reaction, the excessdiamine was removed by evaporation under high vacuum and the free aminogroup in 162a was protected using CbzCl to provide 162b as a white solidafter purification by column chromatography. Alcohol 162b was furtherreacted with compound 4 in the presence of TMSOTf to provide 163a whichwas converted to 163b by removal of the Cbz group using catalytichydrogenation. The pentafluorophenyl (PFP) ester 164 was prepared byreacting triacid 113 (see Example 48) with PFPTFA (3.5 eq) and pyridine(3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reactedwith the amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18.The above method greatly facilitates purification of intermediates andminimizes the formation of byproducts which are formed using theprocedure described in Example 4.

Example 54: Alternate Procedure for Preparation of Compound 18(GalNAc₃-1a and GalNAc₃-3a)

The triPFP ester 164 was prepared from acid 113 using the procedureoutlined in example 53 above and reacted with mono-Boc protected diamineto provide 165 in essentially quantitative yield. The Boc groups wereremoved with hydrochloric acid or trifluoroacetic acid to provide thetriamine which was reacted with the PFP activated acid 166 in thepresence of a suitable base such as DIPEA to provide Compound 18.

The PFP protected Gal-NAc acid 166 was prepared from the correspondingacid by treatment with PFPTFA (1-1.2 eq) and pyridine (1-1.2 eq) in DMF.The precursor acid in turn was prepared from the corresponding alcoholby oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water.The precursor alcohol was prepared from sugar intermediate 4 by reactionwith 1,6-hexanediol (or 1,5-pentanediol or other diol for other nvalues) (2-4 eq) and TMSOTf using conditions described previously inexample 47.

Example 55: Dose-Dependent Study of Oligonucleotides Comprising Either a3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 3, 8 and 9) TargetingSRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the various GalNAc₃ conjugate groups wasattached at either the 3′ or 5′ terminus of the respectiveoligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside(cleavable moiety).

TABLE 26 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′)Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5none 28 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 29^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) ISIS 664078 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -9 29 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -9 _(a) ISIS 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)5/10/5 GalNAc ₃ -3 30 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001GalNAc ₃ -8 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -8 30 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-9 was shown previously in Example 52. The structureof GalNAc₃-3 was shown previously in Example 39. The structure ofGalNAc₃-8 was shown previously in Example 47.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 664078, 661161, 665001 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the liver SRB-1 mRNA levels usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. The resultsbelow are presented as the average percent of SRB-1 mRNA levels for eachtreatment group, normalized to the saline control.

As illustrated in Table 27, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theantisense oligonucleotides comprising the phosphodiester linkedGalNAc₃-1 and GalNAc₃-9 conjugates at the 3′ terminus (ISIS 655861 andISIS 664078) and the GalNAc₃-3 and GalNAc₃-8 conjugates linked at the 5′terminus (ISIS 661161 and ISIS 665001) showed substantial improvement inpotency compared to the unconjugated antisense oligonucleotide (ISIS353382). Furthermore, ISIS 664078, comprising a GalNAc₃-9 conjugate atthe 3′ terminus was essentially equipotent compared to ISIS 655861,which comprises a GalNAc₃-1 conjugate at the 3′ terminus. The 5′conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001,comprising a GalNAc₃-3 or GalNAc₃-9, respectively, had increased potencycompared to the 3′ conjugated antisense oligonucleotides (ISIS 655861and ISIS 664078).

TABLE 27 ASOs containing GalNAc₃-1, 3, 8 or 9 targeting SRB-1 DosageSRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Saline n/a 100 353382 388 none 10 68 30 36 655861 0.5 98 GalNac ₃ -1 (3′) 1.5 76 5 31 15 20664078 0.5 88 GalNac ₃ -9 (3′) 1.5 85 5 46 15 20 661161 0.5 92 GalNac ₃-3 (5′) 1.5 59 5 19 15 11 665001 0.5 100 GalNac ₃ -8 (5′) 1.5 73 5 29 1513

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in the table below.

TABLE 28 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 24 59 0.1 37.52 353382 3 21 66 0.2 34.65 none 10 22 54 0.2 34.230 22 49 0.2 33.72 655861 0.5 25 62 0.2 30.65 GalNac ₃ -1 (3′) 1.5 23 480.2 30.97 5 28 49 0.1 32.92 15 40 97 0.1 31.62 664078 0.5 40 74 0.1 35.3GalNac ₃ -9 (3′) 1.5 47 104 0.1 32.75 5 20 43 0.1 30.62 15 38 92 0.126.2 661161 0.5 101 162 0.1 34.17 GalNac ₃ -3 (5′) 1.5 g 42 100 0.133.37   5 g 23 99 0.1 34.97 15 53 83 0.1 34.8 665001 0.5 28 54 0.1 31.32GalNac ₃ -8 (5′) 1.5 42 75 0.1 32.32 5 24 42 0.1 31.85 15 32 67 0.1 31.

Example 56: Dose-Dependent Study of Oligonucleotides Comprising Either a3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 2, 3, 5, 6, 7 and 10)Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the various GalNAc₃ conjugate groups wasattached at the 5′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety)except for ISIS 655861 which had the GalNAc₃ conjugate group attached atthe 3′ terminus.

TABLE 29 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) MotifConjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5no conjugate 28 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 29 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -1 _(a) ISIS 664507 GalNAc ₃ -2 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -2 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 661161 GalNAc ₃ -3 _(a) - _(o′) A_(do) 5/10/5 GalNAc ₃ -3 30 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666224GalNAc ₃ -5 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5 30^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666961 GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -630 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -730 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -10 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-2_(a) was shown previously in Example 37. Thestructure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-5_(a) was shown previously in Example 49. Thestructure of GalNAc₃-6_(a) was shown previously in Example 51. Thestructure of GalNAc₃-7_(a) was shown previously in Example 48. Thestructure of GalNAc₃-10_(a) was shown previously in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline.Each treatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration to determine the liver SRB-1mRNA levels using real-time PCR and RIBOGREEN® RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.) according to standardprotocols. The results below are presented as the average percent ofSRB-1 mRNA levels for each treatment group, normalized to the salinecontrol.

As illustrated in Table 30, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theconjugated antisense oligonucleotides showed substantial improvement inpotency compared to the unconjugated antisense oligonucleotide (ISIS353382). The 5′ conjugated antisense oligonucleotides showed a slightincrease in potency compared to the 3′ conjugated antisenseoligonucleotide.

TABLE 30 Dosage SRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Salinen/a 100.0 353382 3 96.0 none 10 73.1 30 36.1 655861 0.5 99.4 GalNac ₃ -1(3′) 1.5 81.2 5 33.9 15 15.2 664507 0.5 102.0 GalNac ₃ -2 (5′) 1.5 73.25 31.3 15 10.8 661161 0.5 90.7 GalNac ₃ -3 (5′) 1.5 67.6 5 24.3 15 11.5666224 0.5 96.1 GalNac ₃ -5 (5′) 1.5 61.6 5 25.6 15 11.7 666961 0.5 85.5GalNAc ₃ -6 (5′) 1.5 56.3 5 34.2 15 13.1 666981 0.5 84.7 GalNAc ₃ -7(5′) 1.5 59.9 5 24.9 15 8.5 666881 0.5 100.0 GalNAc ₃ -10 (5′) 1.5 65.85 26.0 15 13.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in Table 31 below.

TABLE 31 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 26 57 0.2 27 353382 3 25 92 0.2 27 none 10 23 40 0.2 25 30 29 540.1 28 655861 0.5 25 71 0.2 34 GalNac ₃ -1 (3′) 1.5 28 60 0.2 26 5 26 630.2 28 15 25 61 0.2 28 664507 0.5 25 62 0.2 25 GalNac ₃ -2 (5′) 1.5 2449 0.2 26 5 21 50 0.2 26 15 59 84 0.1 22 661161 0.5 20 42 0.2 29 GalNac₃ -3 (5′) 1.5 g 37 74 0.2 25   5 g 28 61 0.2 29 15 21 41 0.2 25 6662240.5 34 48 0.2 21 GalNac ₃ -5 (5′) 1.5 23 46 0.2 26 5 24 47 0.2 23 15 3249 0.1 26 666961 0.5 17 63 0.2 26 GalNAc ₃ -6 (5′) 1.5 23 68 0.2 26 5 2566 0.2 26 15 29 107 0.2 28 666981 0.5 24 48 0.2 26 GalNAc ₃ -7 (5′) 1.530 55 0.2 24 5 46 74 0.1 24 15 29 58 0.1 26 666881 0.5 20 65 0.2 27GalNAc ₃ -10 (5′) 1.5 23 59 0.2 24 5 45 70 0.2 26 15 21 57 0.2 24

Example 57: Duration of Action Study of Oligonucleotides Comprising a3′-Conjugate Group Targeting ApoC III In Vivo

Mice were injected once with the doses indicated below and monitoredover the course of 42 days for ApoC-III and plasma triglycerides (PlasmaTG) levels. The study was performed using 3 transgenic mice that expresshuman APOC-III in each group.

TABLE 32 Modified ASO targeting ApoC III SEQ ID ASO Sequence (5′ to 3′)Linkages No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) PS 20 304801 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISISA_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds) PS 21 647535 A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISISA_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds) PO/PS 21 647536 A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

TABLE 33 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (%Saline on Day 1) ASO Dose Target Day 3 Day 7 Day 14 Day 35 Day 42 Saline 0 mg/kg ApoC-III 98 100 100 95 116 ISIS 304801 30 mg/kg ApoC-III 28 3041 65 74 ISIS 647535 10 mg/kg ApoC-III 16 19 25 74 94 ISIS 647536 10mg/kg ApoC-III 18 16 17 35 51 Saline  0 mg/kg Plasma TG 121 130 123 105109 ISIS 304801 30 mg/kg Plasma TG 34 37 50 69 69 ISIS 647535 10 mg/kgPlasma TG 18 14 24 18 71 ISIS 647536 10 mg/kg Plasma TG 21 19 15 32 35

As can be seen in the table above the duration of action increased withaddition of the 3′-conjugate group compared to the unconjugatedoligonucleotide. There was a further increase in the duration of actionfor the conjugated mixed PO/PS oligonucleotide 647536 as compared to theconjugated full PS oligonucleotide 647535.

Example 58: Dose-Dependent Study of Oligonucleotides Comprising a3′-Conjugate Group (Comparison of GalNAc₃-1 and GalNAc4-11) TargetingSRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 wasincluded as an unconjugated standard. Each of the conjugate groups wereattached at the 3′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-11_(a) was shown previously in Example 50.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900, 663748 or with saline. Each treatment group consisted of 4animals. The mice were sacrificed 72 hours following the finaladministration to determine the liver SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 34, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising the phosphodiester linked GalNAc₃-1 andGalNAc4-11 conjugates at the 3′ terminus (ISIS 651900 and ISIS 663748)showed substantial improvement in potency compared to the unconjugatedantisense oligonucleotide (ISIS 440762). The two conjugatedoligonucleotides, GalNAc₃-1 and GalNAc4-11, were equipotent.

TABLE 34 Modified ASO targeting SRB-1 % Saline SEQ ID ASO Sequence(5′ to 3′) Dose mg/kg control No. Saline 100 ISIS 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.673.45 22 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 2 59.66 6 23.50 ISIS 651900T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)0.2 62.75 23 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₃ -1_(a) 0.6 29.14 2 8.61 6 5.62 ISIS 663748 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.263.99 23 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₄ -11 _(a)0.6 33.53 2 7.58 6 5.52

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside; “d”indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o” indicates —O—P(═O)(OH)—. Conjugategroups are in bold.

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in Table 35 below.

TABLE 35 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 30 76 0.2 40 440762 0.60 32 70 0.1 35 none 2 26 57 0.1 35 6 31 480.1 39 651900 0.2 32 115 0.2 39 GalNac ₃ -1 (3′) 0.6 33 61 0.1 35 2 3050 0.1 37 6 34 52 0.1 36 663748 0.2 28 56 0.2 36 GalNac ₄ -11 (3′) 0.634 60 0.1 35 2 44 62 0.1 36 6 38 71 0.1 33

Example 59: Effects of GalNAc₃-1 Conjugated ASOs Targeting FXI In Vivo

The oligonucleotides listed below were tested in a multiple dose studyfor antisense inhibition of FXI in mice. ISIS 404071 was included as anunconjugated standard. Each of the conjugate groups was attached at the3′ terminus of the respective oligonucleotide by a phosphodiester linked2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 36 Modified ASOs targeting FXI SEQ ID ASO Sequence (5′ to 3′)Linkages No. ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 31 404071 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(e) ISIST_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds) PS 32 656172 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIST_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds) PO/PS 32 656173 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(eo) A _(do′) -GalNAc ₃ -1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously twice a week for 3 weeks at the dosage shownbelow with ISIS 404071, 656172, 656173 or with PBS treated control. Eachtreatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration to determine the liver FXI mRNAlevels using real-time PCR and RIBOGREEN® RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.Plasma FXI protein levels were also measured using ELISA. FXI mRNAlevels were determined relative to total RNA (using RIBOGREEN®), priorto normalization to PBS-treated control. The results below are presentedas the average percent of FXI mRNA levels for each treatment group. Thedata was normalized to PBS-treated control and is denoted as “% PBS”.The ED₅₀s were measured using similar methods as described previouslyand are presented below.

TABLE 37 Factor XI mRNA (% Saline) Dose ASO mg/kg % Control ConjugateLinkages Saline 100 none ISIS 3 92 none PS 404071 10 40 30 15 ISIS 0.774 GalNAc ₃ -1 PS 656172 2 33 6 9 ISIS 0.7 49 GalNAc ₃ -1 PO/PS 656173 222 6 1

As illustrated in Table 37, treatment with antisense oligonucleotideslowered FXI mRNA levels in a dose-dependent manner. The oligonucleotidescomprising a 3′-GalNAc₃-1 conjugate group showed substantial improvementin potency compared to the unconjugated antisense oligonucleotide (ISIS404071). Between the two conjugated oligonucleotides an improvement inpotency was further provided by substituting some of the PS linkageswith PO (ISIS 656173).

As illustrated in Table 37a, treatment with antisense oligonucleotideslowered FXI protein levels in a dose-dependent manner. Theoligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showedsubstantial improvement in potency compared to the unconjugatedantisense oligonucleotide (ISIS 404071). Between the two conjugatedoligonucleotides an improvement in potency was further provided bysubstituting some of the PS linkages with PO (ISIS 656173).

TABLE 37a Factor XI protein (% Saline) Dose Protein (% ASO mg/kgControl) Conjugate Linkages Saline 100 none ISIS 3 127 none PS 404071 1032 30 3 ISIS 0.7 70 GalNAc ₃ -1 PS 656172 2 23 6 1 ISIS 0.7 45 GalNAc ₃-1 PO/PS 656173 2 6 6 0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin, total albumin,CRE and BUN were also evaluated. The change in body weights wasevaluated with no significant change from the saline group. ALTs, ASTs,total bilirubin and BUN values are shown in the table below.

TABLE 38 Dosage Total Total ISIS No. mg/kg ALT AST Albumin Bilirubin CREBUN Conjugate Saline 71.8 84.0 3.1 0.2 0.2 22.9 404071 3 152.8 176.0 3.10.3 0.2 23.0 none 10 73.3 121.5 3.0 0.2 0.2 21.4 30 82.5 92.3 3.0 0.20.2 23.0 656172 0.7 62.5 111.5 3.1 0.2 0.2 23.8 GalNac ₃ -1 (3′) 2 33.051.8 2.9 0.2 0.2 22.0 6 65.0 71.5 3.2 0.2 0.2 23.9 656173 0.7 54.8 90.53.0 0.2 0.2 24.9 GalNac ₃ -1 (3′) 2 85.8 71.5 3.2 0.2 0.2 21.0 6 114.0101.8 3.3 0.2 0.2 22.7

Example 60: Effects of Conjugated ASOs Targeting SRB-1 In Vitro

The oligonucleotides listed below were tested in a multiple dose studyfor antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS353382 was included as an unconjugated standard. Each of the conjugategroups were attached at the 3′ or 5′ terminus of the respectiveoligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleosidecleavable moiety.

TABLE 39 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) MotifConjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5none 28 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 29^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) ISIS 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 29 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -1 _(a) ISIS 661161 GalNAc ₃ -3 _(a-o′)A_(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -3 30T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001 GalNAc ₃ -8_(a-o′)A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5GalNAc ₃ -8 30 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664078G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 29^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -9 _(a) ISIS 666961 GalNAc ₃ -6 _(a) ₋ _(o′)A_(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -6 30T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664507 GalNAc ₃ -2 _(a) ₋_(o′)A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)5/10/5 GalNAc ₃ -2 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881GalNAc ₃ -10 _(a) ₋ _(o′)A_(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -10 30^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) ₋ _(o′)A_(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -530 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) ₋ _(o′)A_(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -730 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-3a was shown previously in Example 39. Thestructure of GalNAc₃-8a was shown previously in Example 47. Thestructure of GalNAc₃-9a was shown previously in Example 52. Thestructure of GalNAc₃-6a was shown previously in Example 51. Thestructure of GalNAc₃-2a was shown previously in Example 37. Thestructure of GalNAc₃-10a was shown previously in Example 46. Thestructure of GalNAc₃-5a was shown previously in Example 49. Thestructure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The oligonucleotides listed above were tested in vitro in primary mousehepatocyte cells plated at a density of 25,000 cells per well andtreated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modifiedoligonucleotide. After a treatment period of approximately 16 hours, RNAwas isolated from the cells and mRNA levels were measured byquantitative real-time PCR and the SRB-1 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®.

The IC₅₀ was calculated using standard methods and the results arepresented in Table 40. The results show that, under free uptakeconditions in which no reagents or electroporation techniques are usedto artificially promote entry of the oligonucleotides into cells, theoligonucleotides comprising a GalNAc conjugate were significantly morepotent in hepatocytes than the parent oligonucleotide (ISIS 353382) thatdoes not comprise a GalNAc conjugate.

TABLE 40 Internucleoside SEQ ID ASO IC₅₀ (nM) linkages Conjugate No.ISIS 353382 190^(a ) PS none 28 ISIS 655861  11^(a) PS GalNAc ₃ -1 29ISIS 655862  3 PO/PS GalNAc ₃ -1 29 ISIS 661161  15^(a) PS GalNAc ₃ -330 ISIS 665001 20 PS GalNAc ₃ -8 30 ISIS 664078 55 PS GalNAc ₃ -9 29ISIS 666961  22^(a) PS GalNAc ₃ -6 30 ISIS 664507 30 PS GalNAc ₃ -2 30ISIS 666881 30 PS GalNAc ₃ -10 30 ISIS 666224  30^(a) PS GalNAc ₃ -5 30ISIS 666981 40 PS GalNAc ₃ -7 30 ^(a)Average of multiple runs.

Example 61: Preparation of oligomeric compound 175 comprising GalNAc₃-12

Compound 169 is commercially available. Compound 172 was prepared byaddition of benzyl (perfluorophenyl) glutarate to compound 171. Thebenzyl (perfluorophenyl) glutarate was prepared by adding PFP-TFA andDIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric compound175, comprising a GalNAc₃-12 conjugate group, was prepared from compound174 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-12 (GalNAc₃-12_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups.

In a certain embodiments, the cleavable moiety is —P(═O)(OH)—A_(d)-P(═O)(OH)—. The structure of GalNAc₃-12 (GalNAc₃-12_(a)-CM-) isshown below:

Example 62: Preparation of Oligomeric Compound 180 Comprising GalNAc₃-13

Compound 176 was prepared using the general procedure shown in Example2. Oligomeric compound 180, comprising a GalNAc₃-13 conjugate group, wasprepared from compound 177 using the general procedures illustrated inExample 49. The GalNAc₃ cluster portion of the conjugate groupGalNAc₃-13 (GalNAc₃-13_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. In a certain embodiments, thecleavable moiety is —P(═O)(OH)— A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-13 (GalNAc₃-13_(a)-CM-) is shown below:

Example 63: Preparation of Oligomeric Compound 188 Comprising GalNAc₃-14

Compounds 181 and 185 are commercially available. Oligomeric compound188, comprising a GalNAc₃-14 conjugate group, was prepared from compound187 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-14 (GalNAc₃-14_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is —P(═O)(OH)—A_(d)-P(═O)(OH)—. The structure of GalNAc₃-14 (GalNAc₃-14_(a)-CM-) isshown below:

Example 64: Preparation of Oligomeric Compound 197 Comprising GalNAc₃-15

Compound 189 is commercially available. Compound 195 was prepared usingthe general procedure shown in Example 31. Oligomeric compound 197,comprising a GalNAc₃-15 conjugate group, was prepared from compounds 194and 195 using standard oligonucleotide synthesis procedures. The GalNAc₃cluster portion of the conjugate group GalNAc₃-15 (GalNAc₃-15_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is —P(═O)(OH)—A_(d)-P(═O)(OH)—. The structure of GalNAc₃-15 (GalNAc₃-15_(a)-CM-) isshown below:

Example 65: Dose-Dependent Study of Oligonucleotides Comprising a5′-Conjugate Group (Comparison of GalNAc₃-3, 12, 13, 14, and 15)Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the GalNAc₃ conjugate groups wasattached at the 5′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 41 Modified ASOs targeting SRB-1 SEQ ISIS ID No. Sequences (5′ to3′) Conjugate No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) none 28 661161GalNAc ₃ -3 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-3 30 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671144 GalNAc ₃ -12 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-12 30 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)670061 GalNAc ₃ -13 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-13 30 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671261 GalNAc ₃ -14 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-14 30 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671262 GalNAc ₃ -15 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-15 30 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-12a was shown previously in Example 61. Thestructure of GalNAc₃-13a was shown previously in Example 62. Thestructure of GalNAc₃-14a was shown previously in Example 63. Thestructure of GalNAc₃-15a was shown previously in Example 64.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once or twice at the dosage shown belowwith ISIS 353382, 661161, 671144, 670061, 671261, 671262, or withsaline. Mice that were dosed twice received the second dose three daysafter the first dose. Each treatment group consisted of 4 animals. Themice were sacrificed 72 hours following the final administration todetermine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)according to standard protocols. The results below are presented as theaverage percent of SRB-1 mRNA levels for each treatment group,normalized to the saline control.

As illustrated in Table 42, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. No significantdifferences in target knockdown were observed between animals thatreceived a single dose and animals that received two doses (see ISIS353382 dosages 30 and 2×15 mg/kg; and ISIS 661161 dosages 5 and 2×2.5mg/kg). The antisense oligonucleotides comprising the phosphodiesterlinked GalNAc₃-3, 12, 13, 14, and 15 conjugates showed substantialimprovement in potency compared to the unconjugated antisenseoligonucleotide (ISIS 335382).

TABLE 42 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA (% ISIS No. (mg/kg)Saline) ED₅₀ (mg/kg) Conjugate Saline n/a 100.0 n/a n/a 353382 3 85.022.4 none 10 69.2 30 34.2 2 × 15  36.0 661161 0.5 87.4 2.2 GalNAc₃-3 1.559.0 5 25.6 2 × 2.5 27.5 15 17.4 671144 0.5 101.2 3.4 GalNAc₃-12 1.576.1 5 32.0 15 17.6 670061 0.5 94.8 2.1 GalNAc₃-13 1.5 57.8 5 20.7 1513.3 671261 0.5 110.7 4.1 GalNAc₃-14 1.5 81.9 5 39.8 15 14.1 671262 0.5109.4 9.8 GalNAc₃-15 1.5 99.5 5 69.2 15 36.1

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The changes in body weights were evaluated with nosignificant differences from the saline group (data not shown). ALTs,ASTs, total bilirubin and BUN values are shown in Table 43 below.

TABLE 43 Total Dosage ALT AST Bilirubin BUN ISIS No. (mg/kg) (U/L) (U/L)(mg/dL) (mg/dL) Conjugate Saline n/a 28 60 0.1 39 n/a 353382 3 30 77 0.236 none 10 25 78 0.2 36 30 28 62 0.2 35 2 × 15  22 59 0.2 33 661161 0.539 72 0.2 34 GalNAc₃-3 1.5 26 50 0.2 33 5 41 80 0.2 32 2 × 2.5 24 72 0.228 15 32 69 0.2 36 671144 0.5 25 39 0.2 34 GalNAc₃-12 1.5 26 55 0.2 28 548 82 0.2 34 15 23 46 0.2 32 670061 0.5 27 53 0.2 33 GalNAc₃-13 1.5 2445 0.2 35 5 23 58 0.1 34 15 24 72 0.1 31 671261 0.5 69 99 0.1 33GalNAc₃-14 1.5 34 62 0.1 33 5 43 73 0.1 32 15 32 53 0.2 30 671262 0.5 2451 0.2 29 GalNAc₃-15 1.5 32 62 0.1 31 5 30 76 0.2 32 15 31 64 0.1 32

Example 66: Effect of Various Cleavable Moieties on Antisense InhibitionIn Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Cluster

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugategroups was attached at the 5′ terminus of the respective oligonucleotideby a phosphodiester linked nucleoside (cleavable moiety (CM)).

TABLE 44 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) ₋ _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc_(3-3a) A_(d) 30 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670699 GalNAc ₃-3 _(a) ₋ _(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(d) 33G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)670700 GalNAc ₃ -3 _(a) ₋ _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(e) 30G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)670701 GalNAc ₃ -3 _(a) ₋ _(o′) T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(e) 33G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)671165 GalNAc ₃ -13 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-13a A_(d) 30G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-13a was shown previously in Example 62.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with ISIS661161, 670699, 670700, 670701, 671165, or with saline. Each treatmentgroup consisted of 4 animals. The mice were sacrificed 72 hoursfollowing the final administration to determine the liver SRB-1 mRNAlevels using real-time PCR and RIBOGREEN® RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.The results below are presented as the average percent of SRB-1 mRNAlevels for each treatment group, normalized to the saline control.

As illustrated in Table 45, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising various cleavable moieties all showedsimilar potencies.

TABLE 45 SRB-1 mRNA (% Saline) SRB-1 mRNA GalNAc₃ ISIS No. Dosage(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 87.8GalNAc₃-3a A_(d) 1.5 61.3 5 33.8 15 14.0 670699 0.5 89.4 GalNAc₃-3aT_(d) 1.5 59.4 5 31.3 15 17.1 670700 0.5 79.0 GalNAc₃-3a A_(e) 1.5 63.35 32.8 15 17.9 670701 0.5 79.1 GalNAc₃-3a T_(e) 1.5 59.2 5 35.8 15 17.7671165 0.5 76.4 GalNAc₃-13a A_(d) 1.5 43.2 5 22.6 15 10.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The changes in body weights were evaluated with nosignificant differences from the saline group (data not shown). ALTs,ASTs, total bilirubin and BUN values are shown in Table 46 below.

TABLE 46 Total BUN ISIS Dosage ALT AST Bilirubin (mg/ GalNAc₃ No.(mg/kg) (U/L) (U/L) (mg/dL) dL) Cluster CM Saline n/a 24 64 0.2 31 n/an/a 661161 0.5 25 64 0.2 31 GalNAc₃-3a A_(d) 1.5 24 50 0.2 32 5 26 550.2 28 15 27 52 0.2 31 670699 0.5 42 83 0.2 31 GalNAc₃-3a T_(d) 1.5 3358 0.2 32 5 26 70 0.2 29 15 25 67 0.2 29 670700 0.5 40 74 0.2 27GalNAc₃-3a A_(e) 1.5 23 62 0.2 27 5 24 49 0.2 29 15 25 87 0.1 25 6707010.5 30 77 0.2 27 GalNAc₃-3a T_(e) 1.5 22 55 0.2 30 5 81 101 0.2 25 15 3182 0.2 24 671165 0.5 44 84 0.2 26 GalNAc₃- A_(d) 1.5 47 71 0.1 24 13a 533 91 0.2 26 15 33 56 0.2 29

Example 67: Preparation of Oligomeric Compound 199 Comprising GalNAc₃-16

Oligomeric compound 199, comprising a GalNAc₃-16 conjugate group, isprepared using the general procedures illustrated in Examples 7 and 9.The GalNAc₃ cluster portion of the conjugate group GalNAc₃-16(GalNAc₃-16_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)— A_(d)-P(═O)(OH)—. The structure of GalNAc₃-16(GalNAc₃-16_(a)-CM-) is shown below:

Example 68: Preparation of Oligomeric Compound 200 Comprising GalNAc₃-17

Oligomeric compound 200, comprising a GalNAc₃-17 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-17(GalNAc₃-17_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)— A_(d)-P(═O)(OH)—.The structure of GalNAc₃-17 (GalNAc₃-17_(a)-CM-) is shown below:

Example 69: Preparation of Oligomeric Compound 201 Comprising GalNAc₃-18

Oligomeric compound 201, comprising a GalNAc₃-18 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-18(GalNAc₃-18_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)— A_(d)-P(═O)(OH)—.The structure of GalNAc₃-18 (GalNAc₃-18_(a)-CM-) is shown below:

Example 70: Preparation of Oligomeric Compound 204 Comprising GalNAc₃-19

Oligomeric compound 204, comprising a GalNAc₃-19 conjugate group, wasprepared from compound 64 using the general procedures illustrated inExample 52. The GalNAc₃ cluster portion of the conjugate groupGalNAc₃-19 (GalNAc₃-19_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, thecleavable moiety is —P(═O)(OH)— A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-19 (GalNAc₃-19_(a)-CM-) is shown below:

Example 71: Preparation of Oligomeric Compound 210 Comprising GalNAc₃-20

Compound 205 was prepared by adding PFP-TFA and DIEA to6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which wasprepared by adding triflic anhydride to 6-aminohexanoic acid. Thereaction mixture was heated to 80° C., then lowered to rt. Oligomericcompound 210, comprising a GalNAc₃-20 conjugate group, was prepared fromcompound 208 using the general procedures illustrated in Example 52. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-20(GalNAc₃-20_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)— A_(d)-P(═O)(OH)—. The structure of GalNAc₃-20(GalNAc₃-20_(a)-CM-) is shown below:

Example 72: Preparation of Oligomeric Compound 215 Comprising GalNAc₃-21

Compound 211 is commercially available. Oligomeric compound 215,comprising a GalNAc₃-21 conjugate group, was prepared from compound 213using the general procedures illustrated in Example 52. The GalNAc₃cluster portion of the conjugate group GalNAc₃-21 (GalNAc₃-21_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is —P(═O)(OH)—A_(d)-P(═O)(OH)—. The structure of GalNAc₃-21 (GalNAc₃-21_(a)-CM-) isshown below:

Example 73: Preparation of Oligomeric Compound 221 Comprising GalNAc₃-22

Compound 220 was prepared from compound 219 using diisopropylammoniumtetrazolide. Oligomeric compound 221, comprising a GalNAc₃-21 conjugategroup, is prepared from compound 220 using the general procedureillustrated in Example 52. The GalNAc₃ cluster portion of the conjugategroup GalNAc₃-22 (GalNAc₃-22_(a)) can be combined with any cleavablemoiety to provide a variety of conjugate groups. In certain embodiments,the cleavable moiety is —P(═O)(OH)— A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-22 (GalNAc₃-22_(a)-CM-) is shown below:

Example 74: Effect of Various Cleavable Moieties on Antisense InhibitionIn Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugategroups was attached at the 5′ terminus of the respectiveoligonucleotide.

TABLE 47 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) n/a n/a 28 ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)661161 GalNAc ₃ -3 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 30G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)666904 GalNAc ₃ -3 _(a) ₋ _(o′)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 28G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)675441 GalNAc ₃ -17 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(dsT) _(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-17a A_(d)30 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)675442 GalNAc ₃ -18 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-18a A_(d) 30G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)In all tables, capital letters indicate the nucleobase for eachnucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e”indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-17a was shown previously in Example 68, and thestructure of GalNAc₃-18a was shown in Example 69.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with anoligonucleotide listed in Table 47 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 48, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising a GalNAc conjugate showed similar potenciesand were significantly more potent than the parent oligonucleotidelacking a GalNAc conjugate.

TABLE 48 SRB-1 mRNA (% Saline) SRB-1 mRNA GalNAc₃ ISIS No. Dosage(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 353382 3 79.38n/a n/a 10 68.67 30 40.70 661161 0.5 79.18 GalNAc₃-3a A_(d) 1.5 75.96 530.53 15 12.52 666904 0.5 91.30 GalNAc₃-3a PO 1.5 57.88 5 21.22 15 16.49675441 0.5 76.71 GalNAc₃-17a A_(d) 1.5 63.63 5 29.57 15 13.49 675442 0.595.03 GalNAc₃-18a A_(d) 1.5 60.06 5 31.04 15 19.40

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group (data not shown). ALTs, ASTs,total bilirubin and BUN values are shown in Table 49 below.

TABLE 49 Total BUN ISIS Dosage ALT AST Bilirubin (mg/ GalNAc₃ No.(mg/kg) (U/L) (U/L) (mg/dL) dL) Cluster CM Saline n/a 26 59 0.16 42 n/an/a 353382 3 23 58 0.18 39 n/a n/a 10 28 58 0.16 43 30 20 48 0.12 34661161 0.5 30 47 0.13 35 GalNAc₃-3a A_(d) 1.5 23 53 0.14 37 5 26 48 0.1539 15 32 57 0.15 42 666904 0.5 24 73 0.13 36 GalNAc₃-3a PO 1.5 21 480.12 32 5 19 49 0.14 33 15 20 52 0.15 26 675441 0.5 42 148 0.21 36GalNAc₃- A_(d) 1.5 60 95 0.16 34 17a 5 27 75 0.14 37 15 24 61 0.14 36675442 0.5 26 65 0.15 37 GalNAc₃- A_(d) 1.5 25 64 0.15 43 18a 5 27 690.15 37 15 30 84 0.14 37

Example 75: Pharmacokinetic Analysis of Oligonucleotides Comprising a5′-Conjugate Group

The PK of the ASOs in Tables 41, 44 and 47 above was evaluated usingliver samples that were obtained following the treatment proceduresdescribed in Examples 65, 66, and 74. The liver samples were minced andextracted using standard protocols and analyzed by IP-HPLC-MS alongsidean internal standard. The combined tissue level (μg/g) of allmetabolites was measured by integrating the appropriate UV peaks, andthe tissue level of the full-length ASO missing the conjugate (“parent,”which is Isis No. 353382 in this case) was measured using theappropriate extracted ion chromatograms (EIC).

TABLE 50 PK Analysis in Liver Total Parent Tissue ASO Tissue DosageLevel by Level by GalNAc₃ ISIS No. (mg/kg) UV (μg/g) EIC (μg/g) ClusterCM 353382 3 8.9 8.6 n/a n/a 10 22.4 21.0 30 54.2 44.2 661161 5 32.4 20.7GalNAc₃-3a A_(d) 15 63.2 44.1 671144 5 20.5 19.2 GalNAc₃-12a A_(d) 1548.6 41.5 670061 5 31.6 28.0 GalNAc₃-13a A_(d) 15 67.6 55.5 671261 519.8 16.8 GalNAc₃-14a A_(d) 15 64.7 49.1 671262 5 18.5 7.4 GalNAc₃-15aA_(d) 15 52.3 24.2 670699 5 16.4 10.4 GalNAc₃-3a T_(d) 15 31.5 22.5670700 5 19.3 10.9 GalNAc₃-3a A_(e) 15 38.1 20.0 670701 5 21.8 8.8GalNAc₃-3a T_(e) 15 35.2 16.1 671165 5 27.1 26.5 GalNAc₃-13a A_(d) 1548.3 44.3 666904 5 30.8 24.0 GalNAc₃-3a PO 15 52.6 37.6 675441 5 25.419.0 GalNAc₃-17a A_(d) 15 54.2 42.1 675442 5 22.2 20.7 GalNAc₃-18a A_(d)15 39.6 29.0

The results in Table 50 above show that there were greater liver tissuelevels of the oligonucleotides comprising a GalNAc₃ conjugate group thanof the parent oligonucleotide that does not comprise a GalNAc₃ conjugategroup (ISIS 353382) 72 hours following oligonucleotide administration,particularly when taking into consideration the differences in dosingbetween the oligonucleotides with and without a GalNAc₃ conjugate group.Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising aGalNAc₃ conjugate group was metabolized to the parent compound,indicating that the GalNAc₃ conjugate groups were cleaved from theoligonucleotides.

Example 76: Preparation of Oligomeric Compound 230 Comprising GalNAc₃-23

Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500mL) for 16 hours. The reaction was then evaporated to an oil, dissolvedin EtOAc and washed with water, sat. NaHCO₃, brine, and dried overNa₂SO₄. The ethyl acetate was concentrated to dryness and purified bycolumn chromatography, eluted with EtOAc/hexanes (1:1) followed by 10%methanol in CH₂Cl₂ to give compound 223 as a colorless oil. LCMS and NMRwere consistent with the structure. 10 g (32.86 mmol) of1-Tosyltriethylene glycol (compound 223) was treated with sodium azide(10.68 g, 164.28 mmol) in DMSO (100 mL) at room temperature for 17hours. The reaction mixture was then poured onto water, and extractedwith EtOAc. The organic layer was washed with water three times anddried over Na₂SO₄. The organic layer was concentrated to dryness to give5.3 g of compound 224 (92%). LCMS and NMR were consistent with thestructure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol)and compound 4 (6 g, 18.22 mmol) were treated with 4 A molecular sieves(5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL) underan inert atmosphere. After 14 hours, the reaction was filtered to removethe sieves, and the organic layer was washed with sat. NaHCO₃, water,brine, and dried over Na₂SO₄. The organic layer was concentrated todryness and purified by column chromatography, eluted with a gradient of2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMRwere consistent with the structure. Compound 225 (11.9 g, 23.59 mmol)was hydrogenated in EtOAc/Methanol (4:1, 250 mL) over Pearlman'scatalyst. After 8 hours, the catalyst was removed by filtration and thesolvents removed to dryness to give compound 226. LCMS and NMR wereconsistent with the structure.

In order to generate compound 227, a solution ofnitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's base(10.3 ml, 60.17 mmol) in DMF (100 mL) were treated dropwise withpentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30 minutes,the reaction was poured onto ice water and extracted with EtOAc. Theorganic layer was washed with water, brine, and dried over Na₂SO₄. Theorganic layer was concentrated to dryness and then recrystallized fromheptane to give compound 227 as a white solid. LCMS and NMR wereconsistent with the structure. Compound 227 (1.5 g, 1.93 mmol) andcompound 226 (3.7 g, 7.74 mmol) were stirred at room temperature inacetonitrile (15 mL) for 2 hours. The reaction was then evaporated todryness and purified by column chromatography, eluting with a gradientof 2 to 10% methanol in dichloromethane to give compound 228. LCMS andNMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol)was treated with Raney Nickel (about 2 g wet) in ethanol (100 mL) in anatmosphere of hydrogen. After 12 hours, the catalyst was removed byfiltration and the organic layer was evaporated to a solid that was useddirectly in the next step. LCMS and NMR were consistent with thestructure. This solid (0.87 g, 0.53 mmol) was treated withbenzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA(273.7 μL 1.6 mmol) in DMF (5 mL). After 16 hours, the DMF was removedunder reduced pressure at 65° C. to an oil, and the oil was dissolved indichloromethane. The organic layer was washed with sat. NaHCO₃, brine,and dried over Na₂SO₄. After evaporation of the organic layer, thecompound was purified by column chromatography and eluted with agradient of 2 to 20% methanol in dichloromethane to give the coupledproduct. LCMS and NMR were consistent with the structure. The benzylester was deprotected with Pearlman's catalyst under a hydrogenatmosphere for 1 hour. The catalyst was them removed by filtration andthe solvents removed to dryness to give the acid. LCMS and NMR wereconsistent with the structure. The acid (486 mg, 0.27 mmol) wasdissolved in dry DMF (3 mL). Pyridine (53.61 μl, 0.66 mmol) was addedand the reaction was purged with argon. Pentaflourotriflouro acetate(46.39 μl, 0.4 mmol) was slowly added to the reaction mixture. The colorof the reaction changed from pale yellow to burgundy, and gave off alight smoke which was blown away with a stream of argon. The reactionwas allowed to stir at room temperature for one hour (completion ofreaction was confirmed by LCMS). The solvent was removed under reducedpressure (rotovap) at 70° C. The residue was diluted with DCM and washedwith 1N NaHSO₄, brine, saturated sodium bicarbonate and brine again. Theorganics were dried over Na₂SO₄, filtered, and were concentrated todryness to give 225 mg of compound 229 as a brittle yellow foam. LCMSand NMR were consistent with the structure.

Oligomeric compound 230, comprising a GalNAc₃-23 conjugate group, wasprepared from compound 229 using the general procedure illustrated inExample 46. The GalNAc₃ cluster portion of the GalNAc₃-23 conjugategroup (GalNAc₃-23_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. The structure of GalNAc₃-23(GalNAc₃-23_(a)-CM) is shown below:

Example 77: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice.

TABLE 51 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) ₋ _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 30 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃-3 _(a) ₋ _(o′)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 28G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)673502 GalNAc ₃ -10 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-10a A_(d) 30G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)677844 GalNAc ₃ -9 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-9a A_(d) 30G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)677843 GalNAc ₃ -23 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-23a A_(d) 30G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-1a A_(d) 29 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)677841 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-19a A_(d) 29 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a)677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-20a A_(d) 29 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20 _(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-9a was shown in Example52, GalNAc₃-10a was shown in Example 46, GalNAc₃-19_(a) was shown inExample 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a)was shown in Example 76.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were each injected subcutaneously once at a dosage shown below with anoligonucleotide listed in Table 51 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 52, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 52 SRB-1 mRNA (% Saline) SRB-1 mRNA GalNAc₃ ISIS No. Dosage(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 89.18GalNAc₃-3a A_(d) 1.5 77.02 5 29.10 15 12.64 666904 0.5 93.11 GalNAc₃-3aPO 1.5 55.85 5 21.29 15 13.43 673502 0.5 77.75 GalNAc₃-10a A_(d) 1.541.05 5 19.27 15 14.41 677844 0.5 87.65 GalNAc₃-9a A_(d) 1.5 93.04 540.77 15 16.95 677843 0.5 102.28 GalNAc₃-23a A_(d) 1.5 70.51 5 30.68 1513.26 655861 0.5 79.72 GalNAc₃-1a A_(d) 1.5 55.48 5 26.99 15 17.58677841 0.5 67.43 GalNAc₃-19a A_(d) 1.5 45.13 5 27.02 15 12.41 677842 0.564.13 GalNAc₃-20a A_(d) 1.5 53.56 5 20.47 15 10.23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were also measured using standardprotocols. Total bilirubin and BUN were also evaluated. Changes in bodyweights were evaluated, with no significant change from the saline group(data not shown). ALTs, ASTs, total bilirubin and BUN values are shownin Table 53 below.

TABLE 53 Total BUN ISIS Dosage ALT AST Bilirubin (mg/ GalNAc₃ No.(mg/kg) (U/L) (U/L) (mg/dL) dL) Cluster CM Saline n/a 21 45 0.13 34 n/an/a 661161 0.5 28 51 0.14 39 GalNAc₃-3a A_(d) 1.5 23 42 0.13 39 5 22 590.13 37 15 21 56 0.15 35 666904 0.5 24 56 0.14 37 GalNAc₃-3a PO 1.5 2668 0.15 35 5 23 77 0.14 34 15 24 60 0.13 35 673502 0.5 24 59 0.16 34GalNAc₃- A_(d) 1.5 20 46 0.17 32 10a 5 24 45 0.12 31 15 24 47 0.13 34677844 0.5 25 61 0.14 37 GalNAc₃-9a A_(d) 1.5 23 64 0.17 33 5 25 58 0.1335 15 22 65 0.14 34 677843 0.5 53 53 0.13 35 GalNAc₃- A_(d) 1.5 25 540.13 34 23a 5 21 60 0.15 34 15 22 43 0.12 38 655861 0.5 21 48 0.15 33GalNAc₃-1a A_(d) 1.5 28 54 0.12 35 5 22 60 0.13 36 15 21 55 0.17 30677841 0.5 32 54 0.13 34 GalNAc₃- A_(d) 1.5 24 56 0.14 34 19a 5 23 920.18 31 15 24 58 0.15 31 677842 0.5 23 61 0.15 35 GalNAc₃- A_(d) 1.5 2457 0.14 34 20a 5 41 62 0.15 35 15 24 37 0.14 32

Example 78: Antisense Inhibition In Vivo by Oligonucleotides TargetingAngiotensinogen Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of Angiotensinogen (AGT) in normotensiveSprague Dawley rats.

TABLE 54 Modified ASOs targeting AGT ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 552668 ^(m)C_(es)A_(es)^(m)C_(es)T_(es)G_(es)A_(ds)T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds)^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 34G_(es)A_(es)T_(e) 669509 ^(m)C_(es)A_(es)^(m)C_(es)T_(es)G_(es)A_(ds)T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds)^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es) GalNAc₃-1_(a) A_(d) 35G_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old, male Sprague Dawley rats were each injected subcutaneouslyonce per week at a dosage shown below, for a total of three doses, withan oligonucleotide listed in Table 54 or with PBS. Each treatment groupconsisted of 4 animals. The rats were sacrificed 72 hours following thefinal dose. AGT liver mRNA levels were measured using real-time PCR andRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.) according to standard protocols. AGT plasma protein levels weremeasured using the Total Angiotensinogen ELISA (Catalog # JP27412, IBLInternational, Toronto, ON) with plasma diluted 1:20,000. The resultsbelow are presented as the average percent of AGT mRNA levels in liveror AGT protein levels in plasma for each treatment group, normalized tothe PBS control.

As illustrated in Table 55, treatment with antisense oligonucleotideslowered AGT liver mRNA and plasma protein levels in a dose-dependentmanner, and the oligonucleotide comprising a GalNAc conjugate wassignificantly more potent than the parent oligonucleotide lacking aGalNAc conjugate.

TABLE 55 AGT liver mRNA and plasma protein levels AGT liver AGT plasmaISIS Dosage mRNA protein GalNAc₃ No. (mg/kg) (% PBS) (% PBS) Cluster CMPBS n/a 100 100 n/a n/a 552668 3 95 122 n/a n/a 10 85 97 30 46 79 90 811 669509 0.3 95 70 GalNAc₃-1a A_(d) 1 95 129 3 62 97 10 9 23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in plasma and body weights were also measured attime of sacrifice using standard protocols. The results are shown inTable 56 below.

TABLE 56 Liver transaminase levels and rat body weights Body WeightDosage ALT AST (% of GalNAc₃ ISIS No. (mg/kg) (U/L) (U/L) baseline)Cluster CM PBS n/a 51 81 186 n/a n/a 552668 3 54 93 183 n/a n/a 10 51 93194 30 59 99 182 90 56 78 170 669509 0.3 53 90 190 GalNAc₃-1a A_(d) 1 5193 192 3 48 85 189 10 56 95 189

Example 79: Duration of Action In Vivo of Oligonucleotides TargetingAPOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 57 below were tested in a singledose study for duration of action in mice.

TABLE 57 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es) n/a n/a 20 T_(es)A_(es)T_(e) 647535 A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) GalNAc₃-1a A_(d) 21T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 663083 GalNAc ₃ -3 _(a) ₋_(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 36^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674449GalNAc ₃ -7 _(a) ₋ _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-7a A_(d) 36^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674450GalNAc ₃ -10 _(a) ₋ _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-10a A_(d) 36^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674451GalNAc ₃ -13 _(a) ₋ _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-13a A_(d) 36^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six to eight week old transgenic mice that express human APOC-III wereeach injected subcutaneously once with an oligonucleotide listed inTable 57 or with PBS. Each treatment group consisted of 3 animals. Bloodwas drawn before dosing to determine baseline and at 72 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasmatriglyceride and APOC-III protein levels were measured as described inExample 20. The results below are presented as the average percent ofplasma triglyceride and APOC-III levels for each treatment group,normalized to baseline levels, showing that the oligonucleotidescomprising a GalNAc conjugate group exhibited a longer duration ofaction than the parent oligonucleotide without a conjugate group (ISIS304801) even though the dosage of the parent was three times the dosageof the oligonucleotides comprising a GalNAc conjugate group.

TABLE 58 Plasma triglyceride and APOC-III protein levels in transgenicmice Time point APOC-III Dosage (days post- Triglycerides protein (%GalNAc₃ ISIS No. (mg/kg) dose) (% baseline) baseline) Cluster CM PBS n/a3 97 102 n/a n/a 7 101 98 14 108 98 21 107 107 28 94 91 35 88 90 42 91105 304801 30 3 40 34 n/a n/a 7 41 37 14 50 57 21 50 50 28 57 73 35 6870 42 75 93 647535 10 3 36 37 GalNAc₃-1a A_(d) 7 39 47 14 40 45 21 41 4128 42 62 35 69 69 42 85 102 663083 10 3 24 18 GalNAc₃-3a A_(d) 7 28 2314 25 27 21 28 28 28 37 44 35 55 57 42 60 78 674449 10 3 29 26GalNAc₃-7a A_(d) 7 32 31 14 38 41 21 44 44 28 53 63 35 69 77 42 78 99674450 10 3 33 30 GalNAc₃-10a A_(d) 7 35 34 14 31 34 21 44 44 28 56 6135 68 70 42 83 95 674451 10 3 35 33 GalNAc₃-13a A_(d) 7 24 32 14 40 3421 48 48 28 54 67 35 65 75 42 74 97

Example 80: Antisense Inhibition In Vivo by Oligonucleotides TargetingAlpha-1 Antitrypsin (A1AT) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 59 below were tested in a study fordose-dependent inhibition of A1AT in mice.

TABLE 59 Modified ASOs targeting A1AT ISIS GalNAc₃ SEQ ID No. Sequences(5′ to 3′) Cluster CM No. 476366 A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) n/a n/a 37G_(es)G_(es)A_(e) 656326 A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) GalNAc₃-1aA_(d) 38 G_(es)G_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 678381 GalNAc ₃-3 _(a) ₋ _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)GalNAc₃-3a A_(d) 39 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)678382 GalNAc ₃ -7 _(a) ₋ _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)GalNAc₃-7a A_(d) 39 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)678383 GalNAc ₃ -10 _(a) ₋ _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-10aA_(d) 39 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678384GalNAc ₃ -13 _(a) ₋ _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-13aA_(d) 39 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were each injected subcutaneously once per week at a dosage shown below,for a total of three doses, with an oligonucleotide listed in Table 59or with PBS. Each treatment group consisted of 4 animals. The mice weresacrificed 72 hours following the final administration. A1AT liver mRNAlevels were determined using real-time PCR and RIBOGREEN® RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) accordingto standard protocols. A1AT plasma protein levels were determined usingthe Mouse Alpha 1-Antitrypsin ELISA (catalog #41-A1AMS-E01, Alpco,Salem, NH). The results below are presented as the average percent ofA1AT liver mRNA and plasma protein levels for each treatment group,normalized to the PBS control.

As illustrated in Table 60, treatment with antisense oligonucleotideslowered A1AT liver mRNA and A1AT plasma protein levels in adose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were significantly more potent than the parent (ISIS 476366).

TABLE 60 A1AT liver mRNA and plasma protein levels A1AT A1AT liverplasma ISIS Dosage mRNA protein GalNAc₃ No. (mg/kg) (% PBS) (% PBS)Cluster CM PBS n/a 100 100 n/a n/a 476366 5 86 78 n/a n/a 15 73 61 45 3038 656326 0.6 99 90 GalNAc₃-1a A_(d) 2 61 70 6 15 30 18 6 10 678381 0.6105 90 GalNAc₃-3a A_(d) 2 53 60 6 16 20 18 7 13 678382 0.6 90 79GalNAc₃-7a A_(d) 2 49 57 6 21 27 18 8 11 678383 0.6 94 84 GalNAc₃-10aA_(d) 2 44 53 6 13 24 18 6 10 678384 0.6 106 91 GalNAc₃-13a A_(d) 2 6559 6 26 31 18 11 15

Liver transaminase and BUN levels in plasma were measured at time ofsacrifice using standard protocols. Body weights and organ weights werealso measured. The results are shown in Table 61 below. Body weight isshown as % relative to baseline. Organ weights are shown as % of bodyweight relative to the PBS control group.

TABLE 61 Body Liver Kidney Spleen ISIS Dosage ALT AST BUN weight (%weight (Rel weight (Rel weight (Rel No. (mg/kg) (U/L) (U/L) (mg/dL)baseline) % BW) % BW) % BW) PBS n/a 25 51 37 119 100 100 100 476366 5 3468 35 116 91 98 106 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108123 656326 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 6 3267 39 125 99 97 122 18 46 77 36 116 102 109 101 678381 0.6 26 57 32 11793 109 110 2 26 52 33 121 96 106 125 6 40 78 32 124 92 106 126 18 31 5428 118 94 103 120 678382 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91104 117 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 678383 0.630 67 38 121 91 100 123 2 33 53 33 118 98 102 121 6 32 63 32 117 97 105105 18 36 68 31 118 99 103 108 678384 0.6 36 63 31 118 98 103 98 2 32 6132 119 93 102 114 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104

Example 81: Duration of Action In Vivo of Oligonucleotides TargetingA1AT Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 59 were tested in a single dosestudy for duration of action in mice.

Treatment

Six week old, male C57BL/6 mice were each injected subcutaneously oncewith an oligonucleotide listed in Table 59 or with PBS. Each treatmentgroup consisted of 4 animals. Blood was drawn the day before dosing todetermine baseline and at 5, 12, 19, and 25 days following the dose.Plasma A1AT protein levels were measured via ELISA (see Example 80). Theresults below are presented as the average percent of plasma A1ATprotein levels for each treatment group, normalized to baseline levels.The results show that the oligonucleotides comprising a GalNAc conjugatewere more potent and had longer duration of action than the parentlacking a GalNAc conjugate (ISIS 476366). Furthermore, theoligonucleotides comprising a 5′-GalNAc conjugate (ISIS 678381, 678382,678383, and 678384) were generally even more potent with even longerduration of action than the oligonucleotide comprising a 3′-GalNAcconjugate (ISIS 656326).

TABLE 62 Plasma A1AT protein levels in mice Time point ISIS Dosage (dayspost- A1AT (% GalNAc₃ No. (mg/kg) dose) baseline) Cluster CM PBS n/a 593 n/a n/a 12 93 19 90 25 97 476366 100 5 38 n/a n/a 12 46 19 62 25 77656326 18 5 33 GalNAc₃-1a A_(d) 12 36 19 51 25 72 678381 18 5 21GalNAc₃-3a A_(d) 12 21 19 35 25 48 678382 18 5 21 GalNAc₃-7a A_(d) 12 2119 39 25 60 678383 18 5 24 GalNAc₃-10a A_(d) 12 21 19 45 25 73 678384 185 29 GalNAc₃-13a A_(d) 12 34 19 57 25 76

Example 82: Antisense Inhibition In Vitro by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000cells/well 2 hours prior to treatment. The oligonucleotides listed inTable 63 were added at 2, 10, 50, or 250 nM in Williams E medium andcells were incubated overnight at 37° C. in 5% CO₂. Cells were lysed 16hours following oligonucleotide addition, and total RNA was purifiedusing RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determinedusing real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. IC₅₀ valueswere determined using Prism 4 software (GraphPad). The results show thatoligonucleotides comprising a variety of different GalNAc conjugategroups and a variety of different cleavable moieties are significantlymore potent in an in vitro free uptake experiment than the parentoligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and666841).

TABLE 63 Inhibition of SRB-1 expression in vitro ISIS GalNAc IC₅₀ SEQNo. Sequence (5′ to 3′) Linkages cluster CM (nM) ID No. 353382 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS n/a n/a 250 28^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 29^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) 1_(a) 661161 GalNAc ₃ -3 _(a) ₋ _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃ - A_(d)40 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 661162 GalNAc ₃ -3 _(a) ₋ _(o′) A_(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PSGalNAc₃ - A_(d) 8 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 664078 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 20 29^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -9 _(a) 9_(a) 665001 GalNAc ₃ -8 _(a) ₋ _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 7030 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 8_(a) 666224 GalNAc ₃ -5 _(a) ₋ _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 80 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 5_(a) 666841G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PO/PS n/a n/a >250 28^(m)C_(ds)T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 666881 GalNAc ₃-10 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 30^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 10_(a) 666904 GalNAc ₃ -3 _(a) ₋ _(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) PSGalNAc₃- PO 9 28 A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666924 GalNAc ₃ -3 _(a) ₋ _(o′) T_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- T_(d) 15 33 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666961 GalNAc ₃ -6 _(a) ₋_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)PS GalNAc₃- A_(d) 150 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 6_(a) 666981GalNAc ₃ -7 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 20 30^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 7_(a) 670061 GalNAc ₃ -13 _(a) ₋ _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 30 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 13_(a) 670699 GalNAc ₃ -3 _(a) ₋_(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds)PO/PS GalNAc₃- T_(d) 15 33 ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 670700GalNAc ₃ -3 _(a) ₋ _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(e) 30 30^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T 3_(a) 670701 GalNAc ₃ -3 _(a) ₋ _(o′) T _(eo)G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(e)25 33 ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo)^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 671144 GalNAc ₃ -12 _(a) ₋ _(o′)A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 40 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 12_(a) 671165 GalNAc ₃ -13 _(a)₋ _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 30^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T 13_(a) 671261 GalNAc ₃ -14 _(a) ₋ _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃-A_(d) >250 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 14_(a) 671262 GalNAc ₃ -15 _(a) ₋ _(o′)A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) >250 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 15_(a) 673501GalNAc ₃ -7 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 30 30^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T_(e) 7_(a) 673502 GalNAc ₃ -10 _(a) ₋ _(o′) A_(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PSGalNAc₃- A_(d) 8 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 10_(a) 675441 GalNAc ₃ -17 _(a)₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 30^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 17_(a) 675442 GalNAc ₃ -18 _(a) ₋ _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 20 30 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 18_(a) 677841 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 29^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -19 _(a) 19_(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PSGalNAc₃- A_(d) 30 29 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20 _(a) 20_(a) 677843 GalNAc₃ -23 _(a) ₋ _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40 30^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds) T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 23_(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-5_(a) was shown inExample 49, GalNAc₃-6_(a) was shown in Example 51, GalNAc₃-7_(a) wasshown in Example 48, GalNAc₃-8_(a) was shown in Example 47,GalNAc₃-9_(a) was shown in Example 52, GalNAc₃-10_(a) was shown inExample 46, GalNAc₃-12_(a) was shown in Example 61, GalNAc₃-13_(a) wasshown in Example 62, GalNAc₃-14_(a) was shown in Example 63,GalNAc₃-15_(a) was shown in Example 64, GalNAc₃-17_(a) was shown inExample 68, GalNAc₃-18_(a) was shown in Example 69, GalNAc₃-19_(a) wasshown in Example 70, GalNAc₃-20_(a) was shown in Example 71, andGalNAc₃-23_(a) was shown in Example 76.

Example 83: Antisense Inhibition In Vivo by Oligonucleotides TargetingFactor XI Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 64 below were tested in a study fordose-dependent inhibition of Factor XI in mice.

TABLE 64 Modified oligonucleotides targeting Factor XI ISIS GalNAc SEQNo. Sequence (5′ to 3′) cluster CM ID No. 404071T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 31A_(es)G_(es)G_(e) 656173 T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo) GalNAc₃-1_(a) A_(d) 32 A_(es)G_(es)G_(eo) A_(do′) -GalNAc ₃ -1 _(a) 663086 GalNAc ₃ -3 _(a) _(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-3_(a) A_(d) 40 T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678347 GalNAc ₃ -7 _(a) _(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-7_(a) A_(d) 40 T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678348 GalNAc ₃ -10 _(a) _(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-10_(a) A_(d) 40 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678349 GalNAc ₃ -13 _(a) _(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-13_(a) A_(d) 40 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six to eight week old mice were each injected subcutaneously once perweek at a dosage shown below, for a total of three doses, with anoligonucleotide listed below or with PBS. Each treatment group consistedof 4 animals. The mice were sacrificed 72 hours following the finaldose. Factor XI liver mRNA levels were measured using real-time PCR andnormalized to cyclophilin according to standard protocols. Livertransaminases, BUN, and bilirubin were also measured. The results beloware presented as the average percent for each treatment group,normalized to the PBS control.

As illustrated in Table 65, treatment with antisense oligonucleotideslowered Factor XI liver mRNA in a dose-dependent manner. The resultsshow that the oligonucleotides comprising a GalNAc conjugate were morepotent than the parent lacking a GalNAc conjugate (ISIS 404071).Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS663086, 678347, 678348, and 678349) were even more potent than theoligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 65 Factor XI liver mRNA, liver transaminase, BUN, and bilirubinlevels ISIS Dosage Factor XI ALT AST BUN Bilirubin GalNAc₃ SEQ No.(mg/kg) mRNA (% PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No. PBS n/a100 63 70 21 0.18 n/a n/a 404071 3 65 41 58 21 0.15 n/a 31 10 33 49 5323 0.15 30 17 43 57 22 0.14 656173 0.7 43 90 89 21 0.16 GalNAc₃-1a 32 29 36 58 26 0.17 6 3 50 63 25 0.15 663086 0.7 33 91 169 25 0.16GalNAc₃-3a 40 2 7 38 55 21 0.16 6 1 34 40 23 0.14 678347 0.7 35 28 49 200.14 GalNAc₃-7a 40 2 10 180 149 21 0.18 6 1 44 76 19 0.15 678348 0.7 3943 54 21 0.16 GalNAc₃-10a 40 2 5 38 55 22 0.17 6 2 25 38 20 0.14 6783490.7 34 39 46 20 0.16 GalNAc₃-13a 40 2 8 43 63 21 0.14 6 2 28 41 20 0.14

Example 84: Duration of Action In Vivo of Oligonucleotides TargetingFactor XI Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 64 were tested in a single dosestudy for duration of action in mice.

Treatment

Six to eight week old mice were each injected subcutaneously once withan oligonucleotide listed in Table 64 or with PBS. Each treatment groupconsisted of 4 animals. Blood was drawn by tail bleeds the day beforedosing to determine baseline and at 3, 10, and 17 days following thedose. Plasma Factor XI protein levels were measured by ELISA usingFactor XI capture and biotinylated detection antibodies from R & DSystems, Minneapolis, Minn. (catalog # AF2460 and # BAF2460,respectively) and the OptEJA Reagent Set B (Catalog #550534, BDBiosciences, San Jose, Calif.). The results below are presented as theaverage percent of plasma Factor XI protein levels for each treatmentgroup, normalized to baseline levels. The results show that theoligonucleotides comprising a GalNAc conjugate were more potent withlonger duration of action than the parent lacking a GalNAc conjugate(ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAcconjugate (ISIS 663086, 678347, 678348, and 678349) were even morepotent with an even longer duration of action than the oligonucleotidecomprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 66 Plasma Factor XI protein levels in mice Time point Factor SEQISIS Dosage (days XI (% GalNAc₃ ID No. (mg/kg) post-dose) baseline)Cluster CM No. PBS n/a 3 123 n/a n/a n/a 10 56 17 100 404071 30 3 11 n/an/a 31 10 47 17 52 656173 6 3 1 GalNAc₃-1a A_(d) 32 10 3 17 21 663086 63 1 GalNAc₃-3a A_(d) 40 10 2 17 9 678347 6 3 1 GalNAc₃-7a A_(d) 40 10 117 8 678348 6 3 1 GalNAc₃-10a A_(d) 40 10 1 17 6 678349 6 3 1GalNAc₃-13a A_(d) 40 10 1 17 5

Example 85: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 63 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were each injected subcutaneouslyonce per week at a dosage shown below, for a total of three doses, withan oligonucleotide listed in Table 63 or with saline. Each treatmentgroup consisted of 4 animals. The mice were sacrificed 48 hoursfollowing the final administration to determine the SRB-1 mRNA levelsusing real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. The resultsbelow are presented as the average percent of liver SRB-1 mRNA levelsfor each treatment group, normalized to the saline control.

As illustrated in Tables 67 and 68, treatment with antisenseoligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 67 SRB-1 mRNA in liver SRB-1 mRNA ISIS No. Dosage (mg/kg) (%Saline) GalNAc₃ Cluster CM Saline n/a 100 n/a n/a 655861 0.1 94GalNAc₃-1a A_(d) 0.3 119 1 68 3 32 661161 0.1 120 GalNAc₃-3a A_(d) 0.3107 1 68 3 26 666881 0.1 107 GalNAc₃-10a A_(d) 0.3 107 1 69 3 27 6669810.1 120 GalNAc₃-7a A_(d) 0.3 103 1 54 3 21 670061 0.1 118 GalNAc₃-13aA_(d) 0.3 89 1 52 3 18 677842 0.1 119 GalNAc₃-20a A_(d) 0.3 96 1 65 3 23

TABLE 68 SRB-1 mRNA in liver SRB-1 mRNA ISIS No. Dosage (mg/kg) (%Saline) GalNAc₃ Cluster CM 661161 0.1 107 GalNAc₃-3a A_(d) 0.3 95 1 53 318 677841 0.1 110 GalNAc₃-19a A_(d) 0.3 88 1 52 3 25

Liver transaminase levels, total bilirubin, BUN, and body weights werealso measured using standard protocols. Average values for eachtreatment group are shown in Table 69 below.

TABLE 69 ISIS Dosage ALT AST Bilirubin BUN Body Weight GalNAc₃ No.(mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) Cluster CM Saline n/a19 39 0.17 26 118 n/a n/a 655861 0.1 25 47 0.17 27 114 GalNAc₃-1a A_(d)0.3 29 56 0.15 27 118 1 20 32 0.14 24 112 3 27 54 0.14 24 115 661161 0.135 83 0.13 24 113 GalNAc₃-3a A_(d) 0.3 42 61 0.15 23 117 1 34 60 0.18 22116 3 29 52 0.13 25 117 666881 0.1 30 51 0.15 23 118 GalNAc₃-10a A_(d)0.3 49 82 0.16 25 119 1 23 45 0.14 24 117 3 20 38 0.15 21 112 666981 0.121 41 0.14 22 113 GalNAc₃-7a A_(d) 0.3 29 49 0.16 24 112 1 19 34 0.15 22111 3 77 78 0.18 25 115 670061 0.1 20 63 0.18 24 111 GalNAc₃-13a A_(d)0.3 20 57 0.15 21 115 1 20 35 0.14 20 115 3 27 42 0.12 20 116 677842 0.120 38 0.17 24 114 GalNAc₃-20a A_(d) 0.3 31 46 0.17 21 117 1 22 34 0.1521 119 3 41 57 0.14 23 118

Example 86: Antisense Inhibition In Vivo by Oligonucleotides TargetingTTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 70 below were tested in adose-dependent study for antisense inhibition of human transthyretin(TTR) in transgenic mice that express the human TTR gene.

Treatment

Eight week old TTR transgenic mice were each injected subcutaneouslyonce per week for three weeks, for a total of three doses, with anoligonucleotide and dosage listed in the tables below or with PBS. Eachtreatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration. Tail bleeds were performed atvarious time points throughout the experiment, and plasma TTR protein,ALT, and AST levels were measured and reported in Tables 72-74. Afterthe animals were sacrificed, plasma ALT, AST, and human TTR levels weremeasured, as were body weights, organ weights, and liver human TTR mRNAlevels. TTR protein levels were measured using a clinical analyzer(AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were usedaccording to standard protocols to determine liver human TTR mRNAlevels. The results presented in Tables 71-74 are the average values foreach treatment group. The mRNA levels are the average values relative tothe average for the PBS group. Plasma protein levels are the averagevalues relative to the average value for the PBS group at baseline. Bodyweights are the average percent weight change from baseline untilsacrifice for each individual treatment group. Organ weights shown arenormalized to the animal's body weight, and the average normalized organweight for each treatment group is then presented relative to theaverage normalized organ weight for the PBS group.

In Tables 71-74, “BL” indicates baseline, measurements that were takenjust prior to the first dose. As illustrated in Tables 71 and 72,treatment with antisense oligonucleotides lowered TTR expression levelsin a dose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were more potent than the parent lacking a GalNAc conjugate(ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAcconjugate and mixed PS/PO internucleoside linkages were even more potentthan the oligonucleotide comprising a GalNAc conjugate and full PSlinkages.

TABLE 70 Oligonucleotides targeting human TTR GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 41 A_(es)T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 660261 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS GalNAc₃-1a A_(d) 42A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -1 _(a)682883 GalNAc ₃ -3 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-3a PO 41 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682884 GalNAc ₃ -7 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-7a PO 41 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682885 GalNAc ₃ -10 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/POGalNAc₃-10a PO 41 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682886 GalNAc ₃ -13 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/POGalNAc₃-13a PO 41 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 684057 T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS/PO GalNAc₃-19a A_(d) 42A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19_(a)The legend for Table 72 can be found in Example 74. The structure ofGalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) wasshown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example48. The structure of GalNAc₃-10_(a) was shown in Example 46. Thestructure of GalNAc₃-13_(a) was shown in Example 62. The structure ofGalNAc₃-19_(a) was shown in Example 70.

TABLE 71 Antisense inhibition of human TTR in vivo Plasma TTR TTR IsisDosage mRNA protein GalNAc SEQ No. (mg/kg) (% PBS) (% PBS) cluster CM IDNo. PBS n/a 100 100 n/a n/a 420915 6 99 95 n/a n/a 41 20 48 65 60 18 28660261 0.6 113 87 GalNAc₃-1a A_(d) 42 2 40 56 6 20 27 20 9 11

TABLE 72 Antisense inhibition of human TTR in vivo TTR Plasma TTRprotein (% PBS at BL) SEQ Dosage mRNA Day 17 GalNAc ID Isis No. (mg/kg)(% PBS) BL Day 3 Day 10 (After sac) cluster CM No. PBS n/a 100 100 96 90114 n/a n/a 420915 6 74 106 86 76 83 n/a n/a 41 20 43 102 66 61 58 60 2492 43 29 32 682883 0.6 60 88 73 63 68 GalNAc₃- PO 41 2 18 75 38 23 23 3a6 10 80 35 11 9 682884 0.6 56 88 78 63 67 GalNAc₃- PO 41 2 19 76 44 2523 7a 6 15 82 35 21 24 682885 0.6 60 92 77 68 76 GalNAc₃- PO 41 2 22 9358 32 32 10a 6 17 85 37 25 20 682886 0.6 57 91 70 64 69 GalNAc₃- PO 41 221 89 50 31 30 13a 6 18 102 41 24 27 684057 0.6 53 80 69 56 62 GalNAc₃-A_(d) 42 2 21 92 55 34 30 19a 6 11 82 50 18 13

TABLE 73 Transaminase levels, body weight changes, and relative organweights ALT (U/L) AST (U/L) Body Liver Spleen Kidney SEQ Dosage Day DayDay Day (% (% (% (% ID Isis No. (mg/kg) BL Day 3 10 17 BL Day 3 10 17BL) PBS) PBS) PBS) No. PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100n/a 420915 6 34 33 27 21 64 59 73 47 115 99 89 91 41 20 34 30 28 19 6454 56 42 111 97 83 89 60 34 35 31 24 61 58 71 58 113 102 98 95 6602610.6 33 38 28 26 70 71 63 59 111 96 99 92 42 2 29 32 31 34 61 60 68 61118 100 92 90 6 29 29 28 34 58 59 70 90 114 99 97 95 20 33 32 28 33 6454 68 95 114 101 106 92

TABLE 74 Transaminase levels, body weight changes, and relative organweights ALT (U/L) AST (U/L) Body Liver Spleen Kidney SEQ Dosage Day DayDay Day (% (% (% (% ID Isis No. (mg/kg) BL Day 3 10 17 BL Day 3 10 17BL) PBS) PBS) PBS) No. PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100n/a 420915 6 32 30 34 34 61 71 72 66 102 103 102 105 41 20 41 34 37 3380 76 63 54 106 107 135 101 60 36 30 32 34 58 81 57 60 106 105 104 99682883 0.6 32 35 38 40 53 81 74 76 104 101 112 95 41 2 38 39 42 43 71 8470 77 107 98 116 99 6 35 35 41 38 62 79 103 65 105 103 143 97 682884 0.633 32 35 34 70 74 75 67 101 100 130 99 41 2 31 32 38 38 63 77 66 55 104103 122 100 6 38 32 36 34 65 85 80 62 99 105 129 95 682885 0.6 39 26 3735 63 63 77 59 100 109 109 112 41 2 30 26 38 40 54 56 71 72 102 98 111102 6 27 27 34 35 46 52 56 64 102 98 113 96 682886 0.6 30 40 34 36 58 8754 61 104 99 120 101 41 2 27 26 34 36 51 55 55 69 103 91 105 92 6 40 2834 37 107 54 61 69 109 100 102 99 684057 0.6 35 26 33 39 56 51 51 69 10499 110 102 42 2 33 32 31 40 54 57 56 87 103 100 112 97 6 39 33 35 40 6752 55 92 98 104 121 108

Example 87: Duration of Action In Vivo by Single Doses ofOligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

ISIS numbers 420915 and 660261 (see Table 70) were tested in a singledose study for duration of action in mice. ISIS numbers 420915, 682883,and 682885 (see Table 70) were also tested in a single dose study forduration of action in mice.

Treatment

Eight week old, male transgenic mice that express human TTR were eachinjected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tailbleeds were performed before dosing to determine baseline and at days 3,7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels weremeasured as described in Example 86. The results below are presented asthe average percent of plasma TTR levels for each treatment group,normalized to baseline levels.

TABLE 75 Plasma TTR protein levels Time point ISIS Dosage (days TTR (%GalNAc₃ SEQ No. (mg/kg) post-dose) baseline) Cluster CM ID No. 420915100 3 30 n/a n/a 41 7 23 10 35 17 53 24 75 39 100 660261 13.5 3 27GalNAc₃-1a A_(d) 42 7 21 10 22 17 36 24 48 39 69

Treatment

Female transgenic mice that express human TTR were each injectedsubcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No.682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4animals. Tail bleeds were performed before dosing to determine baselineand at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTRprotein levels were measured as described in Example 86. The resultsbelow are presented as the average percent of plasma TTR levels for eachtreatment group, normalized to baseline levels.

TABLE 76 Plasma TTR protein levels Time point SEQ ISIS Dosage (days TTR(% GalNAc₃ ID No. (mg/kg) post-dose) baseline) Cluster CM No. 420915 1003 48 n/a n/a 41 7 48 10 48 17 66 31 80 682883 10.0 3 45 GalNAc₃-3a PO 417 37 10 38 17 42 31 65 682885 10.0 3 40 GalNAc₃-10a PO 41 7 33 10 34 1740 31 64The results in Tables 75 and 76 show that the oligonucleotidescomprising a GalNAc conjugate are more potent with a longer duration ofaction than the parent oligonucleotide lacking a conjugate (ISIS420915).

Example 88: Splicing Modulation In Vivo by Oligonucleotides TargetingSMN Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 77 were tested for splicingmodulation of human survival of motor neuron (SMN) in mice.

TABLE 77 Modified ASOs targeting SMN ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 387954 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es)^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)n/a n/a 43 G_(e) 699819 GalNAc ₃ -7 _(a) ₋ _(o′)A_(es)T_(es)T_(es)^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es) GalNAc₃-7a PO 43 T_(es)G_(es)^(m)C_(es)T_(es)G_(es)G_(e) 699821 GalNAc ₃ -7 _(a) ₋_(o′)A_(es)T_(eo)T_(eo) ^(m)C_(eo)A_(eo) ^(m)C_(eo)T_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(eo)T_(eo)A_(eo) GalNAc₃-7a PO 43 A_(eo)T_(eo)G_(eo)^(m)C_(eo)T_(es)G_(es)G_(e) 700000 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es)^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)GalNAc₃-1a A_(d) 44 G_(eo) A _(do′) -GalNAc ₃ -1 _(a) 703421X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG n/a n/a 43 703422 GalNAc ₃ -7_(b)-X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG GalNAc₃-7b n/a 43The structure of GalNAc₃-7_(a) was shown previously in Example 48. “X”indicates a 5′ primary amine generated by Gene Tools (Philomath, Oreg.),and GalNAc₃-7_(b) indicates the structure of GalNAc₃-7_(a) lacking the—NH—C₆—O portion of the linker as shown below:

ISIS numbers 703421 and 703422 are morphlino oligonucleotides, whereineach nucleotide of the two oligonucleotides is a morpholino nucleotide.

Treatment

Six week old transgenic mice that express human SMN were injectedsubcutaneously once with an oligonucleotide listed in Table 78 or withsaline. Each treatment group consisted of 2 males and 2 females. Themice were sacrificed 3 days following the dose to determine the liverhuman SMN mRNA levels both with and without exon 7 using real-time PCRaccording to standard protocols. Total RNA was measured using Ribogreenreagent. The SMN mRNA levels were normalized to total mRNA, and furthernormalized to the averages for the saline treatment group. The resultingaverage ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7are shown in Table 78. The results show that fully modifiedoligonucleotides that modulate splicing and comprise a GalNAc conjugateare significantly more potent in altering splicing in the liver than theparent oligonucleotides lacking a GlaNAc conjugate. Furthermore, thistrend is maintained for multiple modification chemistries, including2′-MOE and morpholino modified oligonucleotides.

TABLE 78 Effect of oligonucleotides targeting human SMN in vivo ISISDose GalNAc₃ SEQ No. (mg/kg) +Exon 7/−Exon 7 Cluster CM ID No. Salinen/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 43 387954 288 5.00 n/a n/a43 699819 32 7.84 GalNAc₃-7a PO 43 699821 32 7.22 GalNAc₃-7a PO 43700000 32 6.91 GalNAc₃-1a A_(d) 44 703421 32 1.27 n/a n/a 43 703422 324.12 GalNAc₃-7b n/a 43

Example 89: Antisense Inhibition In Vivo by Oligonucleotides TargetingApolipoprotein A (Apo(a)) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 79 below were tested in a study fordose-dependent inhibition of Apo(a) in transgenic mice.

TABLE 79 Modified ASOs targeting Apo(a) ISIS GalNAc₃ SEQ ID No.Sequences (5′ to 3′) Cluster CM No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es)^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds) n/a n/a 53 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257GalNAc ₃ -7 _(a) _(-o′) T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 53 T_(ds)G_(ds)^(m)C_(ds) T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor,Me.) were each injected subcutaneously once per week at a dosage shownbelow, for a total of six doses, with an oligonucleotide listed in Table79 or with PBS. Each treatment group consisted of 3-4 animals. Tailbleeds were performed the day before the first dose and weekly followingeach dose to determine plasma Apo(a) protein levels. The mice weresacrificed two days following the final administration. Apo(a) livermRNA levels were determined using real-time PCR and RIBOGREEN® RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) accordingto standard protocols. Apo(a) plasma protein levels were determinedusing ELISA, and liver transaminase levels were determined. The mRNA andplasma protein results in Table 80 are presented as the treatment groupaverage percent relative to the PBS treated group. Plasma protein levelswere further normalized to the baseline (BL) value for the PBS group.Average absolute transaminase levels and body weights (% relative tobaseline averages) are reported in Table 81.

As illustrated in Table 80, treatment with the oligonucleotides loweredApo(a) liver mRNA and plasma protein levels in a dose-dependent manner.Furthermore, the oligonucleotide comprising the GalNAc conjugate wassignificantly more potent with a longer duration of action than theparent oligonucleotide lacking a GalNAc conjugate. As illustrated inTable 81, transaminase levels and body weights were unaffected by theoligonucleotides, indicating that the oligonucleotides were welltolerated.

TABLE 80 Apo(a) liver mRNA and plasma protein levels ISIS Dosage Apo(a)mRNA Apo(a) plasma protein (% PBS) No. (mg/kg) (% PBS) BL Week 1 Week 2Week 3 Week 4 Week 5 Week 6 PBS n/a 100 100 120 119 113 88 121 97 4943723 80 84 89 91 98 87 87 79 10 30 87 72 76 71 57 59 46 30 5 92 54 28 10 79 7 681257 0.3 75 79 76 89 98 71 94 78 1 19 79 88 66 60 54 32 24 3 2 8252 17 7 4 6 5 10 2 79 17 6 3 2 4 5

TABLE 81 Dosage ALT AST Body weight ISIS No. (mg/kg) (U/L) (U/L) (%baseline) PBS n/a 37 54 103 494372 3 28 68 106 10 22 55 102 30 19 48 103681257 0.3 30 80 104 1 26 47 105 3 29 62 102 10 21 52 107

Example 90: Antisense Inhibition In Vivo by Oligonucleotides TargetingTTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 82 below were tested in adose-dependent study for antisense inhibition of human transthyretin(TTR) in transgenic mice that express the human TTR gene.

Treatment

TTR transgenic mice were each injected subcutaneously once per week forthree weeks, for a total of three doses, with an oligonucleotide anddosage listed in Table 83 or with PBS. Each treatment group consisted of4 animals. Prior to the first dose, a tail bleed was performed todetermine plasma TTR protein levels at baseline (BL). The mice weresacrificed 72 hours following the final administration. TTR proteinlevels were measured using a clinical analyzer (AU480, Beckman Coulter,CA). Real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) were used according to standard protocols todetermine liver human TTR mRNA levels. The results presented in Table 83are the average values for each treatment group. The mRNA levels are theaverage values relative to the average for the PBS group. Plasma proteinlevels are the average values relative to the average value for the PBSgroup at baseline. “BL” indicates baseline, measurements that were takenjust prior to the first dose. As illustrated in Table 83, treatment withantisense oligonucleotides lowered TTR expression levels in adose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were more potent than the parent lacking a GalNAc conjugate(ISIS 420915), and oligonucleotides comprising a phosphodiester ordeoxyadenosine cleavable moiety showed significant improvements inpotency compared to the parent lacking a conjugate (see ISIS numbers682883 and 666943 vs 420915 and see Examples 86 and 87).

TABLE 82 Oligonucleotides targeting human TTR GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 41 A_(es)T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682883 GalNAc ₃ -3 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-3a PO 41 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 666943 GalNAc ₃ -3 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-3aA_(d) 45 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682887 GalNAc ₃-7 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-7aA_(d) 45 ^(m)C_(ds)A_(ds)T_(as)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682888 GalNAc ₃ -10 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-10aA_(d) 45 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682889 GalNAc ₃ -13 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-13aA_(d) 45 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e)The legend for Table 82 can be found in Example 74. The structure ofGalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a)was shown in Example 48. The structure of GalNAc₃-10_(a) was shown inExample 46. The structure of GalNAc₃-13_(a) was shown in Example 62.

TABLE 83 Antisense inhibition of human TTR in vivo Dosage TTR mRNA TTRprotein GalNAc Isis No. (mg/kg) (% PBS) (% BL) cluster CM PBS n/a 100124 n/a n/a 420915 6 69 114 n/a n/a 20 71 86 60 21 36 682883 0.6 61 73GalNAc₃-3a PO 2 23 36 6 18 23 666943 0.6 74 93 GalNAc₃-3a A_(d) 2 33 576 17 22 682887 0.6 60 97 GalNAc₃-7a A_(d) 2 36 49 6 12 19 682888 0.6 6592 GalNAc₃-10a A_(d) 2 32 46 6 17 22 682889 0.6 72 74 GalNAc₃-13a A_(d)2 38 45 6 16 18

Example 91: Antisense Inhibition In Vivo by Oligonucleotides TargetingFactor VII Comprising a GalNAc₃ Conjugate in Non-Human Primates

Oligonucleotides listed in Table 84 below were tested in a non-terminal,dose escalation study for antisense inhibition of Factor VII in monkeys.

Treatment

Non-naïve monkeys were each injected subcutaneously on days 0, 15, and29 with escalating doses of an oligonucleotide listed in Table 84 orwith PBS. Each treatment group consisted of 4 males and 1 female. Priorto the first dose and at various time points thereafter, blood drawswere performed to determine plasma Factor VII protein levels. Factor VIIprotein levels were measured by ELISA. The results presented in Table 85are the average values for each treatment group relative to the averagevalue for the PBS group at baseline (BL), the measurements taken justprior to the first dose. As illustrated in Table 85, treatment withantisense oligonucleotides lowered Factor VII expression levels in adose-dependent manner, and the oligonucleotide comprising the GalNAcconjugate was significantly more potent in monkeys compared to theoligonucleotide lacking a GalNAc conjugate.

TABLE 84 Oligonucleotides targeting Factor VII GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 407935 A_(es)T_(es)G_(es)^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds) PS n/a n/a 46 T_(es) ^(m)C_(es)T_(es)G_(es)A_(e) 686892GalNAc ₃ -10 _(a-o′) A _(es)T_(es)G_(es)^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) PS GalNAc₃-10a PO 46A_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)T_(es)G_(es)A_(e)

The legend for Table 84 can be found in Example 74. The structure ofGalNAc₃-10_(a) was shown in Example 46.

TABLE 85 Factor VII plasma protein levels ISIS No. Day Dose (mg/kg)Factor VII (% BL) 407935 0 n/a 100 15 10 87 22 n/a 92 29 30 77 36 n/a 4643 n/a 43 686892 0  3 100 15 10 56 22 n/a 29 29 30 19 36 n/a 15 43 n/a11

Example 92: Antisense Inhibition in Primary Hepatocytes by AntisenseOligonucleotides Targeting Apo-CIII Comprising a GalNAc₃ Conjugate

Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cellsper well, and the oligonucleotides listed in Table 86, targeting mouseApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or333.33 nM or 1.00 μM. After incubation with the oligonucleotides for 24hours, the cells were lysed and total RNA was purified using RNeasy(Qiagen). ApoC-III mRNA levels were determined using real-time PCR andRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) accordingto standard protocols. IC₅₀ values were determined using Prism 4software (GraphPad). The results show that regardless of whether thecleavable moiety was a phosphodiester or a phosphodiester-linkeddeoxyadensoine, the oligonucleotides comprising a GalNAc conjugate weresignificantly more potent than the parent oligonucleotide lacking aconjugate.

TABLE 86 Inhibition of mouse APOC-III expression in mouse primaryhepatocytes ISIS IC₅₀ SEQ No. Sequence (5′ to 3′) CM (nM) ID No. 440670^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) n/a 13.20 47 661180^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) A_(d) 1.40 48 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc₃ -1 _(a) 680771 GalNAc ₃ -3 _(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 0.70 47 A_(es)G_(es) ^(m)C_(es)A_(e) 680772 GalNAc ₃ -7_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 1.70 47 A_(es)G_(es) ^(m)C_(es)A_(e) 680773 GalNAc ₃ -10_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 2.00 47 A_(es)G_(es) ^(m)C_(es)A_(e) 680774 GalNAc ₃ -13_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 1.50 47 A_(es)G_(es) ^(m)C_(es)A_(e) 681272 GalNAc ₃ -3_(a-o′) ^(m)C_(es)A_(eo)G_(eo)^(m)C_(eo)T_(eo)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(eo) PO <0.46 47 A_(eo)G_(es) ^(m)C_(es)A_(e) 681273 GalNAc ₃ -3_(a) - _(o′) A _(do) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)A_(d) 1.10 49 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 683733^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) A_(d) 2.50 48 A_(es)G_(es) ^(m)C_(es)A_(eo)A_(do′)-GalNAc ₃-19 _(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-13_(a) wasshown in Example 62, and GalNAc₃-19_(a) was shown in Example 70.

Example 93: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising Mixed Wings and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 87 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 87 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequence(5′ to 3′) Cluster CM ID No. 449093 T_(ks)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds) A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) n/a n/a 50 699806 GalNAc ₃-3 _(a) - _(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-3a PO 50 T_(ds)T_(ks)^(m)C_(ks) ^(m)C_(k) 699807 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds)^(m)C_(ds) GalNAc₃-7a PO 50 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699809GalNAc ₃ -7 _(a) - _(o′) T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds) A_(ds)T_(ds) G_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 50T_(ds)T_(es) ^(m)C_(es) ^(m)C_(e) 699811 GalNAc ₃ -7 _(a) -_(o′)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 50 T_(ds)T_(ks) ^(m)C_(ks)^(m)C_(k) 699813 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ds)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds)^(m)C_(ds) GalNAc₃-7a PO 50 T_(ds)T_(ks) ^(m)C_(ds) ^(m)C_(k) 699815GalNAc ₃ -7 _(a) - _(o′)T_(es)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 50T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(e)The structure of GalNAc₃-3_(a) was shown previously in Example 39, andthe structure of GalNAc₃-7a was shown previously in Example 48.Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o”indicates phosphodiester internucleoside linkages (PO). Superscript “m”indicates 5-methylcytosines.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with anoligonucleotide listed in Table 87 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration. Liver SRB-1 mRNA levels were measured usingreal-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNAlevels according to standard protocols. The results are presented as theaverage percent of SRB-1 mRNA levels for each treatment group relativeto the saline control group. As illustrated in Table 88, treatment withantisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependentmanner, and the gapmer oligonucleotides comprising a GalNAc conjugateand having wings that were either full cEt or mixed sugar modificationswere significantly more potent than the parent oligonucleotide lacking aconjugate and comprising full cEt modified wings.

Body weights, liver transaminases, total bilirubin, and BUN were alsomeasured, and the average values for each treatment group are shown inTable 88. Body weight is shown as the average percent body weightrelative to the baseline body weight (% BL) measured just prior to theoligonucleotide dose.

TABLE 88 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and bodyweights SRB-1 Body ISIS Dosage mRNA ALT AST weight No. (mg/kg) (% PBS)(U/L) (U/L) Bil BUN (% BL) PBS n/a 100 31 84 0.15 28 102 449093 1 111 1848 0.17 31 104 3 94 20 43 0.15 26 103 10 36 19 50 0.12 29 104 699806 0.1114 23 58 0.13 26 107 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104699807 0.1 121 19 41 0.14 25 100 0.3 73 23 56 0.13 26 105 1 24 22 690.14 25 102 699809 0.1 125 23 57 0.14 26 104 0.3 70 20 49 0.10 25 105 133 34 62 0.17 25 107 699811 0.1 123 48 77 0.14 24 106 0.3 94 20 45 0.1325 101 1 66 57 104 0.14 24 107 699813 0.1 95 20 58 0.13 28 104 0.3 98 2261 0.17 28 105 1 49 19 47 0.11 27 106 699815 0.1 93 30 79 0.17 25 1050.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106

Example 94: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising 2′-Sugar Modifications and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 89 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 89 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequence(5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) n/a n/a 28 T_(es)T_(e)700989 G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)n/a n/a 51 U_(ms)U_(m) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-3a PO 28^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 700991 GalNAc ₃-7 _(a) - _(o)′G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds) GalNAc₃-7a PO 51 A_(ds)^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)U_(ms)U_(m)Subscript “m” indicates a 2′-O-methyl modified nucleoside. See Example74 for complete table legend. The structure of GalNAc₃-3_(a) was shownpreviously in Example 39, and the structure of GalNAc₃-7a was shownpreviously in Example 48.

Treatment

The study was completed using the protocol described in Example 93.Results are shown in Table 90 below and show that both the 2′-MOE and2′-OMe modified oligonucleotides comprising a GalNAc conjugate weresignificantly more potent than the respective parent oligonucleotideslacking a conjugate. The results of the body weights, livertransaminases, total bilirubin, and BUN measurements indicated that thecompounds were all well tolerated.

TABLE 90 SRB-1 mRNA ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a100 353382 5 116 15 58 45 27 700989 5 120 15 92 45 46 666904 1 98 3 4510 17 700991 1 118 3 63 10 14

Example 95: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising Bicyclic Nucleosides and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 91 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 91 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/an/a 22 666905 GalNAc ₃ -3 _(a) - _(o′)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-3_(a) PO 22 699782 GalNAc ₃ -7_(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)GalNAc₃-7_(a) PO 22 699783 GalNAc ₃ -3 _(a) - _(o′)T_(ls)^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(l) GalNAc₃-3_(a) PO 22 653621 T_(ls)^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(lo) A _(do′)-GalNAc ₃ -1 _(a)GalNAc₃-1_(a) A_(d) 23 439879 T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(d) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) n/an/a 22 699789 GalNAc ₃ -3 _(a) - _(o′)T_(gs)^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d) G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) GalNAc₃-3_(a) PO 22Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicatesa locked nucleoside comprising a 2′-O—CH₂-4′ bridge. See the Example 74table legend for other abbreviations. The structure of GalNAc₃-1_(a) wasshown previously in Example 9, the structure of GalNAc₃-3_(a) was shownpreviously in Example 39, and the structure of GalNAc₃-7a was shownpreviously in Example 48.

Treatment

The study was completed using the protocol described in Example 93.Results are shown in Table 92 below and show that oligonucleotidescomprising a GalNAc conjugate and various bicyclic nucleosidemodifications were significantly more potent than the parentoligonucleotide lacking a conjugate and comprising bicyclic nucleosidemodifications. Furthermore, the oligonucleotide comprising a GalNAcconjugate and fluoro-HNA modifications was significantly more potentthan the parent lacking a conjugate and comprising fluoro-HNAmodifications. The results of the body weights, liver transaminases,total bilirubin, and BUN measurements indicated that the compounds wereall well tolerated.

TABLE 92 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and bodyweights ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 440762 1104 3 65 10 35 666905 0.1 105 0.3 56 1 18 699782 0.1 93 0.3 63 1 15699783 0.1 105 0.3 53 1 12 653621 0.1 109 0.3 82 1 27 439879 1 96 3 7710 37 699789 0.1 82 0.3 69 1 26

Example 96: Plasma Protein Binding of Antisense OligonucleotidesComprising a GalNAc₃ Conjugate Group

Oligonucleotides listed in Table 57 targeting ApoC-III andoligonucleotides in Table 93 targeting Apo(a) were tested in anultra-filtration assay in order to assess plasma protein binding.

TABLE 93 Modified oligonucleotides targeting Apo(a) ISIS GalNAc₃ SEQ No.Sequences (5′ to 3′) Cluster CM ID No 494372 T_(es)G_(es)^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) n/a n/a 53 T_(es) ^(m)C_(e) 693401T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es) n/a n/a 53 T_(es) ^(m)C_(e) 681251GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)GalNAc₃-7_(a) PO 53 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)GalNAc₃-7_(a) PO 53 T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)See the Example 74 for table legend. The structure of GalNAc₃-7a wasshown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regeneratedcellulose membrane, Millipore, Bedford, Mass.) were pre-conditioned with300 μL of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, thenwith 3004 of a 300 μg/mL solution of a control oligonucleotide in H₂Oand centrifuged at 2000 g for 16 minutes. In order to assessnon-specific binding to the filters of each test oligonucleotide fromTables 57 and 93 to be used in the studies, 300 μL of a 250 ng/mLsolution of oligonucleotide in H₂O at pH 7.4 was placed in thepre-conditioned filters and centrifuged at 2000 g for 16 minutes. Theunfiltered and filtered samples were analyzed by an ELISA assay todetermine the oligonucleotide concentrations. Three replicates were usedto obtain an average concentration for each sample. The averageconcentration of the filtered sample relative to the unfiltered sampleis used to determine the percent of oligonucleotide that is recoveredthrough the filter in the absence of plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from normal, drug-freehuman volunteers, cynomolgus monkeys, and CD-1 mice, were purchased fromBioreclamation LLC (Westbury, N.Y.). The test oligonucleotides wereadded to 1.2 mL aliquots of plasma at two concentrations (5 and 150μg/mL). An aliquot (300 μL) of each spiked plasma sample was placed in apre-conditioned filter unit and incubated at 37° C. for 30 minutes,immediately followed by centrifugation at 2000 g for 16 minutes.Aliquots of filtered and unfiltered spiked plasma samples were analyzedby an ELISA to determine the oligonucleotide concentration in eachsample. Three replicates per concentration were used to determine theaverage percentage of bound and unbound oligonucleotide in each sample.The average concentration of the filtered sample relative to theconcentration of the unfiltered sample is used to determine the percentof oligonucleotide in the plasma that is not bound to plasma proteins (%unbound). The final unbound oligonucleotide values are corrected fornon-specific binding by dividing the % unbound by the % recovery foreach oligonucleotide. The final % bound oligonucleotide values aredetermined by subtracting the final % unbound values from 100. Theresults are shown in Table 94 for the two concentrations ofoligonucleotide tested (5 and 150 μg/mL) in each species of plasma. Theresults show that GalNAc conjugate groups do not have a significantimpact on plasma protein binding. Furthermore, oligonucleotides withfull PS internucleoside linkages and mixed PO/PS linkages both bindplasma proteins, and those with full PS linkages bind plasma proteins toa somewhat greater extent than those with mixed PO/PS linkages.

TABLE 94 Percent of modified oligonucleotide bound to plasma proteinsISIS Human plasma Monkey plasma Mouse plasma No. 5 μg/mL 150 μg/mL 5μg/mL 150 μg/mL 5 μg/mL 150 μg/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.689.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.094.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.693.7 95.6 92.7

Example 97: Modified Oligonucleotides Targeting TTR Comprising a GalNAc₃Conjugate Group

The oligonucleotides shown in Table 95 comprising a GalNAc conjugatewere designed to target TTR.

TABLE 95 Modified oligonucleotides targeting TTR GalNAc₃ SEQ ID ISIS No.Sequences (5′ to 3′) Cluster CM No 666941 GalNAc ₃ -3 _(a-o′) A _(do)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-3 A_(d) 45 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 666942 T_(es) ^(m)C_(eo) T_(eo)T_(eo) G_(eo) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds)G_(ds) A_(ds) A_(ds) GalNAc₃-1 A_(d) 42 A_(eo) T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -3 _(a) 682876 GalNAc ₃ -3_(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-3 PO 41 G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682877 GalNAc ₃ -7_(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-7 PO 41 G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682878 GalNAc ₃ -10_(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-10 PO 41 T_(ds) G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682879 GalNAc ₃ -13_(a-o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-13 PO 41 T_(ds) G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682880 GalNAc ₃ -7 _(a-o′)A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7 A_(d) 45 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds)A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682881 GalNAc ₃ -10_(a-o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds)T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-10 A_(d) 45 A_(ds) T_(ds) G_(ds) A_(ds)A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682882 GalNAc ₃ -13_(a-o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds)T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-13 A_(d) 45 A_(ds) T_(ds) G_(ds) A_(ds)A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 684056 T_(es)^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds)A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) GalNAc₃-19 A_(d) 42 A_(es) T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19 _(a)The legend for Table 95 can be found in Example 74. The structure ofGalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) wasshown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example48. The structure of GalNAc₃-10_(a) was shown in Example 46. Thestructure of GalNAc₃-13_(a) was shown in Example 62. The structure ofGalNAc₃-19_(a) was shown in Example 70.

Example 98: Evaluation of Pro-Inflammatory Effects of OligonucleotidesComprising a GalNAc Conjugate in hPMBC Assay

The oligonucleotides listed in Table 96 and were tested forpro-inflammatory effects in an hPMBC assay as described in Examples 23and 24. (See Tables 17, 70, 82, and 95 for descriptions of theoligonucleotides.) ISIS 353512 is a high responder used as a positivecontrol, and the other oligonucleotides are described in Tables 70, 82,and 95. The results shown in Table 96 were obtained using blood from onevolunteer donor. The results show that the oligonucleotides comprisingmixed PO/PS internucleoside linkages produced significantly lowerpro-inflammatory responses compared to the same oligonucleotides havingfull PS linkages. Furthermore, the GalNAc conjugate group did not have asignificant effect in this assay.

TABLE 96 ISIS No. E_(max)/EC₅₀ GalNAc₃ cluster Linkages CM 353512 3630n/a PS n/a 420915 802 n/a PS n/a 682881 1311 GalNAc₃-10 PS A_(d) 6828880.26 GalNAc₃-10 PO/PS A_(d) 684057 1.03 GalNAc₃-19 PO/PS A_(d)

Example 99: Binding Affinities of Oligonucleotides Comprising a GalNAcConjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides listed in Table 97 (seeTable 63 for descriptions of the oligonucleotides) for theasialoglycoprotein receptor were tested in a competitive receptorbinding assay. The competitor ligand, α1-acid glycoprotein (AGP), wasincubated in 50 mM sodium acetate buffer (pH 5) with 1 Uneuraminidase-agarose for 16 hours at 3TC, and >90% desilylation wasconfirmed by either sialic acid assay or size exclusion chromatography(SEC). Iodine monochloride was used to iodinate the AGP according to theprocedure by Atsma et al. (see J Lipid Res. 1991 January; 32(1):173-81.)In this method, desialylated al-acid glycoprotein (de-AGP) was added to10 mM iodine chloride, Na¹²⁵I, and 1 M glycine in 0.25 M NaOH. Afterincubation for 10 minutes at room temperature, ¹²⁵I-labeled de-AGP wasseparated from free ¹²⁵I by concentrating the mixture twice utilizing a3 KDMWCO spin column. The protein was tested for labeling efficiency andpurity on a HPLC system equipped with an Agilent SEC-3 column (7.8×300mm) and a β-RAM counter. Competition experiments utilizing ¹²⁵I-labeledde-AGP and various GalNAc-cluster containing ASOs were performed asfollows. Human HepG2 cells (10⁶ cells/ml) were plated on 6-well platesin 2 ml of appropriate growth media. MEM media supplemented with 10%fetal bovine serum (FBS), 2 mM L-Glutamine and 10 mM HEPES was used.Cells were incubated 16-20 hours @ 37° C. with 5% and 10% CO₂respectively. Cells were washed with media without FBS prior to theexperiment. Cells were incubated for 30 min @37° C. with 1 mlcompetition mix containing appropriate growth media with 2% FBS, 10⁻⁸M¹²⁵I-labeled de-AGP and GalNAc-cluster containing ASOs atconcentrations ranging from 10⁻¹¹ to 10⁻⁵ M. Non-specific binding wasdetermined in the presence of 10⁻² M GalNAc sugar. Cells were washedtwice with media without FBS to remove unbound ¹²⁵I-labeled de-AGP andcompetitor GalNAc ASO. Cells were lysed using Qiagen's RLT buffercontaining 1% β-mercaptoethanol. Lysates were transferred to roundbottom assay tubes after a brief 10 min freeze/thaw cycle and assayed ona γ-counter. Non-specific binding was subtracted before dividing ¹²⁵Iprotein counts by the value of the lowest GalNAc-ASO concentrationcounts. The inhibition curves were fitted according to a single sitecompetition binding equation using a nonlinear regression algorithm tocalculate the binding affinities (K_(D)'s).

The results in Table 97 were obtained from experiments performed on fivedifferent days. Results for oligonucleotides marked with superscript “a”are the average of experiments run on two different days. The resultsshow that the oligonucleotides comprising a GalNAc conjugate group onthe 5′-end bound the asialoglycoprotein receptor on human HepG2 cellswith 1.5 to 16-fold greater affinity than the oligonucleotidescomprising a GalNAc conjugate group on the 3′-end.

TABLE 97 Asialoglycoprotein receptor binding assay resultsOligonucleotide end to which GalNAc ISIS No. GalNAc conjugate conjugateis attached K_(D) (nM) 661161^(a) GalNAc₃-3 5′ 3.7 666881^(a) GalNAc₃-105′ 7.6 666981 GalNAc₃-7 5′ 6.0 670061 GalNAc₃-13 5′ 7.4 655861^(a)GalNAc₃-1 3′ 11.6 677841^(a) GalNAc₃-19 3′ 60.8

Example 100: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 98a below were tested in a singledose study for duration of action in mice.

TABLE 98a Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 681251 GalNAc ₃ -7 _(a)-_(o′)T_(es)G_(es)^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)GalNAc₃-7a PO 53 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es)^(m)C_(e) 681257 GalNAc ₃ -7 _(a)-_(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo)^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 53T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Female transgenic mice that express human Apo(a) were each injectedsubcutaneously once per week, for a total of 6 doses, with anoligonucleotide and dosage listed in Table 98b or with PBS. Eachtreatment group consisted of 3 animals. Blood was drawn the day beforedosing to determine baseline levels of Apo(a) protein in plasma and at72 hours, 1 week, and 2 weeks following the first dose. Additional blooddraws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following thefirst dose. Plasma Apo(a) protein levels were measured using an ELISA.The results in Table 98b are presented as the average percent of plasmaApo(a) protein levels for each treatment group, normalized to baselinelevels (% BL), The results show that the oligonucleotides comprising aGalNAc conjugate group exhibited potent reduction in Apo(a) expression.This potent effect was observed for the oligonucleotide that comprisesfull PS internucleoside linkages and the oligonucleotide that comprisesmixed PO and PS linkages.

TABLE 98b Apo(a) plasma protein levels Apo(a) at Apo(a) at Apo(a) at 72hours 1 week 3 weeks ISIS No. Dosage (mg/kg) (% BL) (% BL) (% BL) PBSn/a 116 104 107 681251 0.3 97 108 93 1.0 85 77 57 3.0 54 49 11 10.0 2315 4 681257 0.3 114 138 104 1.0 91 98 54 3.0 69 40 6 10.0 30 21 4

Example 101: Antisense Inhibition by Oligonucleotides Comprising aGalNAc Cluster Linked Via a Stable Moiety

The oligonucleotides listed in Table 99 were tested for inhibition ofmouse APOC-III expression in vivo. C57Bl/6 mice were each injectedsubcutaneously once with an oligonucleotide listed in Table 99 or withPBS. Each treatment group consisted of 4 animals. Each mouse treatedwith ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mousetreated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. TheGalNAc conjugate group of ISIS 696847 is linked via a stable moiety, aphosphorothioate linkage instead of a readily cleavable phosphodiestercontaining linkage. The animals were sacrificed 72 hours after the dose.Liver APOC-III mRNA levels were measured using real-time PCR. APOC-IIImRNA levels were normalized to cyclophilin mRNA levels according tostandard protocols. The results are presented in Table 99 as the averagepercent of APOC-III mRNA levels for each treatment group relative to thesaline control group. The results show that the oligonucleotidescomprising a GalNAc conjugate group were significantly more potent thanthe oligonucleotide lacking a conjugate group. Furthermore, theoligonucleotide comprising a GalNAc conjugate group linked to theoligonucleotide via a cleavable moiety (ISIS 680772) was even morepotent than the oligonucleotide comprising a GalNAc conjugate grouplinked to the oligonucleotide via a stable moiety (ISIS 696847).

TABLE 99 Modified oligonucleotides targeting mouse APOC-III APOC-III SEQISIS Dosage mRNA (% ID No. Sequences (5′ to 3′) CM (mg/kg) PBS) No.440670 ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) n/a 2 92 47G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 6 86 2059 60 37 680772 GalNAc ₃ -7 _(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds) PO 0.6 79 47T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es)^(m)C_(es)A_(e) 2 58 6 31 20 13 696847 GalNAc ₃ -7 _(a-s′)^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds) n/a (PS)0.6 83 47 T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es) A_(es)G_(es)^(m)C_(es)A_(e) 2 73 6 40 20 28The structure of GalNAc₃-7_(a) was shown in Example 48.

Example 102: Distribution in Liver of Antisense OligonucleotidesComprising a GalNAc Conjugate

The liver distribution of ISIS 353382 (see Table 23) that does notcomprise a GalNAc conjugate and ISIS 655861 (see Table 23) that doescomprise a GalNAc conjugate was evaluated. Male balb/c mice weresubcutaneously injected once with ISIS 353382 or 655861 at a dosagelisted in Table 100. Each treatment group consisted of 3 animals exceptfor the 18 mg/kg group for ISIS 655861, which consisted of 2 animals.The animals were sacrificed 48 hours following the dose to determine theliver distribution of the oligonucleotides. In order to measure thenumber of antisense oligonucleotide molecules per cell, a Ruthenium (II)tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to anoligonucleotide probe used to detect the antisense oligonucleotides. Theresults presented in Table 100 are the average concentrations ofoligonucleotide for each treatment group in units of millions ofoligonucleotide molecules per cell. The results show that at equivalentdoses, the oligonucleotide comprising a GalNAc conjugate was present athigher concentrations in the total liver and in hepatocytes than theoligonucleotide that does not comprise a GalNAc conjugate. Furthermore,the oligonucleotide comprising a GalNAc conjugate was present at lowerconcentrations in non-parenchymal liver cells than the oligonucleotidethat does not comprise a GalNAc conjugate. And while the concentrationsof ISIS 655861 in hepatocytes and non-parenchymal liver cells weresimilar per cell, the liver is approximately 80% hepatocytes by volume.Thus, the majority of the ISIS 655861 oligonucleotide that was presentin the liver was found in hepatocytes, whereas the majority of the ISIS353382 oligonucleotide that was present in the liver was found innon-parenchymal liver cells.

TABLE 100 Concentration in Concentration Concentration innon-parenchymal in whole liver hepatocytes liver cells ISIS Dosage(molecules * (molecules * (molecules * No. (mg/kg) 10{circumflex over( )}6 per cell) 10{circumflex over ( )}6 per cell) 10{circumflex over( )}6 per cell) 353382 3 9.7 1.2 37.2 10 17.3 4.5 34.0 20 23.6 6.6 65.630 29.1 11.7 80.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 655861 0.5 2.62.9 3.2 1 6.2 7.0 8.8 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.377.1

Example 103: Duration of Action In Vivo of Oligonucleotides TargetingAPOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 101 below were tested in a singledose study for duration of action in mice.

TABLE 101 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No.Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 20T_(es)A_(es)T_(e) 663084 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)GalNAc₃-3a A_(d) 36 ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e) 679241 A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo) GalNAc₃-19a A_(d) 21 T_(es)A_(es)T_(eo) A _(do′)-GalNAc ₃ -19 _(a)The structure of GalNAc₃-3_(a) was shown in Example 39, andGalNAc₃-19_(a) was shown in Example 70.

Treatment

Female transgenic mice that express human APOC-III were each injectedsubcutaneously once with an oligonucleotide listed in Table 101 or withPBS. Each treatment group consisted of 3 animals. Blood was drawn beforedosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 daysfollowing the dose. Plasma triglyceride and APOC-III protein levels weremeasured as described in Example 20. The results in Table 102 arepresented as the average percent of plasma triglyceride and APOC-IIIlevels for each treatment group, normalized to baseline levels. Acomparison of the results in Table 58 of example 79 with the results inTable 102 below show that oligonucleotides comprising a mixture ofphosphodiester and phosphorothioate internucleoside linkages exhibitedincreased duration of action than equivalent oligonucleotides comprisingonly phosphorothioate internucleoside linkages.

TABLE 102 Plasma triglyceride and APOC-III protein levels in transgenicmice Time point APOC-III Dosage (days post- Triglycerides protein (%GalNAc₃ ISIS No. (mg/kg) dose) (% baseline) baseline) Cluster CM PBS n/a3 96 101 n/a n/a 7 88 98 14 91 103 21 69 92 28 83 81 35 65 86 42 72 88304801 30 3 42 46 n/a n/a 7 42 51 14 59 69 21 67 81 28 79 76 35 72 95 4282 92 663084 10 3 35 28 GalNAc₃-3a A_(d) 7 23 24 14 23 26 21 23 29 28 3022 35 32 36 42 37 47 679241 10 3 38 30 GalNAc₃- A_(d) 7 31 28 19a 14 3022 21 36 34 28 48 34 35 50 45 42 72 64

Example 104: Synthesis of Oligonucleotides Comprising a 5′-GalNAc2Conjugate

Compound 120 is commercially available, and the synthesis of compound126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU(0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF(10 mL. and N,N-diisopropylethylamine (1.75 mL, 10.1 mmol) were added.After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol)was added to the reaction. After 3 h, the reaction mixture was pouredinto 100 mL of 1 M NaHSO₄ and extracted with 2×50 mL ethyl acetate.Organic layers were combined and washed with 3×40 mL sat NaHCO₃ and 2×brine, dried with Na₂SO₄, filtered and concentrated. The product waspurified by silica gel column chromatography (DCM:EA:Hex, 1:1:1) toyield compound 231. LCMS and NMR were consistent with the structure.Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10mL) and trifluoracetic acid (10 mL) was added. After stirring at roomtemperature for 2 h, the reaction mixture was concentrated under reducedpressure and co-evaporated with toluene (3×10 mL). The residue was driedunder reduced pressure to yield compound 232 as the trifuloracetatesalt. The synthesis of compound 166 is described in Example 54. Compound166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution ofcompound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) andN,N-diisopropylethylamine (1.55 mL) was added. The reaction was stirredat room temperature for 30 minutes, then poured into water (80 mL) andthe aqueous layer was extracted with EtOAc (2×100 mL). The organic phasewas separated and washed with sat. aqueous NaHCO₃ (3×80 mL), 1 M NaHSO₄(3×80 mL) and brine (2×80 mL), then dried (Na₂SO₄), filtered, andconcentrated. The residue was purified by silica gel columnchromatography to yield compound 233. LCMS and NMR were consistent withthe structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved inmethanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt% Pd/C, wet, 0.07 g) was added, and the reaction mixture was stirredunder hydrogen atmosphere for 3 h. The reaction mixture was filteredthrough a pad of Celite and concentrated to yield the carboxylic acid.The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolvedin DMF (3.2 mL). To this N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol)and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring atroom temperature the reaction mixture was poured into water (40 mL) andextracted with EtOAc (2×50 mL). A standard work-up was completed asdescribed above to yield compound 234. LCMS and NMR were consistent withthe structure. Oligonucleotide 235 was prepared using the generalprocedure described in Example 46. The GalNAc₂ cluster portion(GalNAc₂-24_(a)) of the conjugate group GalNAc₂-24 can be combined withany cleavable moiety present on the oligonucleotide to provide a varietyof conjugate groups. The structure of GalNAc₂-24 (GalNAc₂-24_(a)-CM) isshown below:

Example 105: Synthesis of Oligonucleotides Comprising a GalNAc1-25Conjugate

The synthesis of compound 166 is described in Example 54.Oligonucleotide 236 was prepared using the general procedure describedin Example 46.

Alternatively, oligonucleotide 236 was synthesized using the schemeshown below, and compound 238 was used to form the oligonucleotide 236using procedures described in Example 10.

The GalNAc₁ cluster portion (GalNAc₁-25_(a)) of the conjugate groupGalNAc₁-25 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-25 (GalNAc₁-25_(a)-CM) is shown below:

Example 106: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a 5′-GalNAc2 or a 5′-GalNAc₃ Conjugate

Oligonucleotides listed in Tables 103 and 104 were tested indose-dependent studies for antisense inhibition of SRB-1 in mice.

Treatment

Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No.440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222,or 708561; or with saline. Each treatment group consisted of 4 animals.The mice were sacrificed 72 hours following the final administration.Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNAlevels were normalized to cyclophilin mRNA levels according to standardprotocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in adose-dependent manner, and the ED₅₀ results are presented in Tables 103and 104. Although previous studies showed that trivalentGalNAc-conjugated oligonucleotides were significantly more potent thandivalent GalNAc-conjugated oligonucleotides, which were in turnsignificantly more potent than monovalent GalNAc conjugatedoligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem., Vol.16, 5216-5231 (2008)), treatment with antisense oligonucleotidescomprising monovalent, divalent, and trivalent GalNAc clusters loweredSRB-1 mRNA levels with similar potencies as shown in Tables 103 and 104.

TABLE 103 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No.Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 4.7 22 686221 GalNAc ₂ -24 _(a) -_(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₂-24_(a) 0.39 26^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 686222 GalNAc ₃ -13 _(a) - _(o′) A_(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-13_(a) 0.41 26^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)See Example 93 for table legend. The structure of GalNAc₃-13a was shownin Example 62, and the structure of GalNAc₂-24a was shown in Example104.

TABLE 104 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No.Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 5 22 708561 GalNAc ₁ -25 _(a) -_(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₁-25_(a) 0.4 22^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)See Example 93 for table legend. The structure of GalNAc₁-25a was shownin Example 105.

The concentrations of the oligonucleotides in Tables 103 and 104 inliver were also assessed, using procedures described in Example 75. Theresults shown in Tables 104a and 104b below are the average totalantisense oligonucleotide tissues levels for each treatment group, asmeasured by UV in units of μg oligonucleotide per gram of liver tissue.The results show that the oligonucleotides comprising a GalNAc conjugategroup accumulated in the liver at significantly higher levels than thesame dose of the oligonucleotide lacking a GalNAc conjugate group.Furthermore, the antisense oligonucleotides comprising one, two, orthree GalNAc ligands in their respective conjugate groups allaccumulated in the liver at similar levels. This result is surprising inview of the Khorev et al. literature reference cited above and isconsistent with the activity data shown in Tables 103 and 104 above.

TABLE 104a Liver concentrations of oligonucleotides comprising a GalNAc₂or GalNAc₃ conjugate group Dosage [Antisense GalNAc ISIS No. (mg/kg)oligonucleotide] (μg/g) cluster CM 440762 2 2.1 n/a n/a 7 13.1 20 31.1686221 0.2 0.9 GalNAc₂-24_(a) A_(d) 0.6 2.7 2 12.0 6 26.5 686222 0.2 0.5GalNAc₃-13_(a) A_(d) 0.6 1.6 2 11.6 6 19.8

TABLE 104b Liver concentrations of oligonucleotides comprising a GalNAc₁conjugate group Dosage [Antisense ISIS No. (mg/kg) oligonucleotide](μg/g) GalNAc cluster CM 440762 2 2.3 n/a n/a 7 8.9 20 23.7 708561 0.20.4 GalNAc₁-25_(a) PO 0.6 1.1 2 5.9 6 23.7 20 53.9

Example 107: Synthesis of Oligonucleotides Comprising a GalNAc1-26 orGalNAc1-27 Conjugate

Oligonucleotide 239 is synthesized via coupling of compound 47 (seeExample 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. Theresulting amide containing compound is phosphitylated, then added to the5′-end of an oligonucleotide using procedures described in Example 10.The GalNAc₁ cluster portion (GalNAc₁-26_(a)) of the conjugate groupGalNAc₁-26 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-26 (GalNAc₁-26_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of anoligonucleotide, the amide formed from the reaction of compounds 47 and64 is added to a solid support using procedures described in Example 7.The oligonucleotide synthesis is then completed using proceduresdescribed in Example 9 in order to form oligonucleotide 240.

The GalNAc₁ cluster portion (GalNAc₁-27_(a)) of the conjugate groupGalNAc₁-27 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-27 (GalNAc₁-27_(a)-CM) is shown below:

Example 108: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 105 below were tested in a singledose study in mice.

TABLE 105 Modified ASOs targeting APO(a) ISIS SEQ No. Sequences (5′ to3′) GalNAc₃ Cluster CM ID No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es)^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds) n/a n/a 53 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681251GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 53 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681255 GalNAc ₃ -3_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-3a PO 53 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681256 GalNAc ₃ -10_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-10a PO 53 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 53 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681258 GalNAc ₃ -13_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-13a PO 53 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681260 T_(es)G_(eo)^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo) GalNAc₃-19a A_(d) 52 T_(es)T_(es)^(m)C_(eo) A _(do′) -GalNAc ₃ -19The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Male transgenic mice that express human Apo(a) were each injectedsubcutaneously once with an oligonucleotide and dosage listed in Table106 or with PBS. Each treatment group consisted of 4 animals. Blood wasdrawn the day before dosing to determine baseline levels of Apo(a)protein in plasma and at 1 week following the first dose. Additionalblood draws will occur weekly for approximately 8 weeks. Plasma Apo(a)protein levels were measured using an ELISA. The results in Table 106are presented as the average percent of plasma Apo(a) protein levels foreach treatment group, normalized to baseline levels (% BL), The resultsshow that the antisense oligonucleotides reduced Apo(a) proteinexpression. Furthermore, the oligonucleotides comprising a GalNAcconjugate group exhibited even more potent reduction in Apo(a)expression than the oligonucleotide that does not comprise a conjugategroup.

TABLE 106 Apo(a) plasma protein levels Apo(a) at 1 week ISIS No. Dosage(mg/kg) (% BL) PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 68125610 17 681257 10 24 681258 10 22 681260 10 26

Example 109: Synthesis of Oligonucleotides Comprising a GalNAc1-28 orGalNAc1-29 Conjugate

Oligonucleotide 241 is synthesized using procedures similar to thosedescribed in Example 71 to form the phosphoramidite intermediate,followed by procedures described in Example 10 to synthesize theoligonucleotide. The GalNAc₁ cluster portion (GalNAc₁-28_(a)) of theconjugate group GalNAc₁-28 can be combined with any cleavable moietypresent on the oligonucleotide to provide a variety of conjugate groups.The structure of GalNAc1-28 (GalNAc₁-28_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of anoligonucleotide, procedures similar to those described in Example 71 areused to form the hydroxyl intermediate, which is then added to the solidsupport using procedures described in Example 7. The oligonucleotidesynthesis is then completed using procedures described in Example 9 inorder to form oligonucleotide 242.

The GalNAc₁ cluster portion (GalNAc₁-29_(a)) of the conjugate groupGalNAc₁-29 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc1-29 (GalNAc₁-29_(a)-CM) is shown below:

Example 110: Synthesis of Oligonucleotides Comprising a GalNAc₁-30Conjugate

Oligonucleotide 246 comprising a GalNAc₁-30 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₁ cluster portion (GalNAc₁-30_(a)) of theconjugate group GalNAc₁-30 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, Y is partof the cleavable moiety. In certain embodiments, Y is part of a stablemoiety, and the cleavable moiety is present on the oligonucleotide. Thestructure of GalNAc₁-30_(a) is shown below:

Example 111: Synthesis of Oligonucleotides Comprising a GalNAc₂-31 orGalNAc₂-32 Conjugate

Oligonucleotide 250 comprising a GalNAc₂-31 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc2 cluster portion (GalNAc₂-31_(a)) of theconjugate group GalNAc₂-31 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, theY-containing group directly adjacent to the 5′-end of theoligonucleotide is part of the cleavable moiety. In certain embodiments,the Y-containing group directly adjacent to the 5′-end of theoligonucleotide is part of a stable moiety, and the cleavable moiety ispresent on the oligonucleotide. The structure of GalNAc₂-31_(a) is shownbelow:

The synthesis of an oligonucleotide comprising a GalNAc₂-32 conjugate isshown below.

Oligonucleotide 252 comprising a GalNAc₂-32 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₂ cluster portion (GalNAc₂-32_(a)) of theconjugate group GalNAc₂-32 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, theY-containing group directly adjacent to the 5′-end of theoligonucleotide is part of the cleavable moiety. In certain embodiments,the Y-containing group directly adjacent to the 5′-end of theoligonucleotide is part of a stable moiety, and the cleavable moiety ispresent on the oligonucleotide. The structure of GalNAc₂-32_(a) is shownbelow:

Example 112: Modified Oligonucleotides Comprising a GalNAc₁ Conjugate

The oligonucleotides in Table 107 targeting SRB-1 were synthesized witha GalNAc₁ conjugate group in order to further test the potency ofoligonucleotides comprising conjugate groups that contain one GalNAcligand.

TABLE 107 GalNAc SEQ ISIS No. Sequence (5′ to 3′) cluster CM ID NO.711461 GalNAc ₁ -25 _(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es)^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-25_(a) A_(d)30 T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) T_(e) 711462 GalNAc ₁ -25 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es)^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-25_(a)PO 28 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) T_(e) 711463 GalNAc ₁ -25 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo)^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-25_(a) PO 28T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es) T_(e) 711465 GalNAc ₁ -26 _(a-o′) A _(do) G_(es) ^(m)C_(es)T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds)GalNAc₁-26_(a) A_(d) 30 T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es)^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711466 GalNAc ₁ -26 _(a-o′)G_(es)^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds)A_(ds) T_(ds) GalNAc₁-26_(a) PO 28 G_(ds) A_(ds) ^(m)C_(ds) T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711467 GalNAc ₁ -26_(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds)^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-26_(a) PO 28 G_(ds) A_(ds) ^(m)C_(ds)T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(e) 711468 GalNAc ₁ -28_(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds)T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-28_(a) A_(d) 30 T_(ds) G_(ds) A_(ds)^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711469GalNAc ₁ -28 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds)G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-28_(a) PO 28 G_(ds)A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e)711470 GalNAc ₁ -28 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo)A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-28_(a) PO 28G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es)T_(e) 713844 G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds)T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds)GalNAc₁-27_(a) PO 28 T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(eo′-) GalNAc₁ -27 _(a) 713845 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds)G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds)GalNAc₁-27_(a) PO 28 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo′-) GalNAc₁ -27 _(a) 713846 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds)G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds)GalNAc₁-27_(a) A_(d) 29 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo) A_(do′-) GalNAc ₁ -27 _(a) 713847 G_(es) ^(m)C_(es) T_(es) T_(es)^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds)^(m)C_(ds) T_(ds) GalNAc₁-29_(a) PO 28 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) T_(eo′-) GalNAc ₁ -29 _(a) 713848 G_(es) ^(m)C_(eo) T_(eo) T_(eo)^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds)^(m)C_(ds) T_(ds) GalNAc₁-29_(a) PO 28 T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es) T_(eo′-) GalNAc ₁ -29 _(a) 713849 G_(es) ^(m)C_(es) T_(es) T_(es)^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds)^(m)C_(ds) T_(ds) GalNAc₁-29_(a) A_(d) 29 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) T_(eo) A _(do′-) GalNAc ₁ -29 _(a) 713850 G_(es) ^(m)C_(eo)T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds)G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) A_(d) 29 T_(eo)^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo) A _(do′-) GalNAc ₁ -29 _(a)

1. A compound comprising a modified oligonucleotide and a conjugategroup, wherein the modified oligonucleotide is 10-30 linked nucleosidesin length and has the nucleobase sequence of SEQ ID NO: 55, wherein theconjugate group comprises:

and wherein the conjugate group is linked to the modifiedoligonucleotide at the 5′ end of the modified oligonucleotide. 2-40.(canceled)
 41. The compound of claim 1, wherein the modifiedoligonucleotide comprises at least one modified sugar.
 42. The compoundof claim 41, wherein the modified sugar is a bicyclic sugar.
 43. Thecompound of claim 42, wherein the bicyclic sugar is selected from thegroup consisting of: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)₂—O-2′ (ENA); and4′-CH(CH₃)—O-2′ (cEt).
 44. The compound of claim 42, wherein themodified sugar is 2′-O-methoxyethyl.
 45. The compound of claim 1,wherein the modified oligonucleotide comprises at least one modifiednucleobase.
 46. The compound of claim 45, wherein the modifiednucleobase is a 5-methylcytosine. 47-54. (canceled)
 55. The compound ofclaim 1, wherein the compound is single-stranded.
 56. The compound ofclaim 1, wherein the compound is double-stranded.
 57. The compound ofclaim 1, wherein the modified oligonucleotide comprises at least onemodified internucleoside linkage.
 58. The compound of claim 57, whereinthe modified internucleoside linkage is a phosphorothioateinternucleoside linkage.
 59. The compound of claim 57, wherein themodified oligonucleotide comprises at least one phosphodiesterinternucleoside linkage. 60-213. (canceled)
 214. A compositioncomprising the compound of claim 1 or salt thereof and at least one of apharmaceutically acceptable carrier or diluent.
 215. A prodrugcomprising the compound of claim
 1. 216. A method comprisingadministering to an animal the compound or composition of claim
 1. 217.The method of claim 216, wherein the animal is a human.
 218. The methodof claim 216, comprising co-administering the compound or compositionand a second agent.
 219. (canceled)